Rickettsia bellii in ticks Amblyomma varium Koch, 1844, from birds in Peru

Amazonian birds were caught and examined for the presence of ectoparasites in the Allpahuayo Mishana National Reserve near Iquitos, Peru, from 13 to 16 August 2011. A total of 40 birds representing 16 species were examined. Two birds (5%) were infested with 2 larvae of Amblyomma varium Koch, 1844, and one nymph of A. calcaratum Neumann, 1899. The 2 larvae of A. varium were infected with Rickettsia bellii. This is the first report of R. bellii in A. varium and also the first record of this rickettsia in Peru. In addition, an immature A. calcaratum is reported from Peru for the first time. (c) 2012 Elsevier GmbH. All rights reserved.

A surrogate life cycle of Amblyomma ovale Koch, 1844

As ticks spend most of their time off-host, the environment is a major selective force of these parasites. In fact, human impact on landscapes has favored a minority of tick species which became well-known pests. However, this is an ongoing process and novel pests may arise. We herein report a surrogate life cycle of a neotropical tick species. Amblyomma ovate, and which may be related to an increased risk of human rickettsiosis. Under natural conditions, adults of this tick species feed on carnivores and exhibit non-nidicolous ambush behavior, whereas larvae and nymphs feed on small rodents and birds. In an anthropized spot within an Atlantic rainforest reserve of Brazil, an A. ovate population exhibited a nidicolous behavior with all 3 tick stages feeding on the dog. This dog's infestation was outstandingly high, and it displayed the highest anti-Rickettsia titers and harbored Rickettsia-infected ticks. (c) 2012 Elsevier GmbH. All rights reserved.

Brazilian spotted fever: Real-time PCR for diagnosis of fatal cases

Suspicion of Brazilian spotted fever (BSF) should occur in endemic regions upon surveillance of the acute febrile icteric hemorrhagic syndrome (AFIHS). However, limitations associated with currently available laboratory tests pose a challenge to early diagnosis, especially in fatal cases. Two real-time PCR (qPCR) protocols were evaluated to diagnose BSF in 110 fatal AFIHS cases, collected in BSF-endemic regions in 2009-2010. Of these, 24 were positive and 86 negative by indirect immunofluorescence (IFA) assay (cutoff IgG and/or IgM >= 128). DNA from these samples was used in the qPCR protocols: one to detect Rickettsia spp. (Citrate synthase gene) and another to determine spotted fever group (SFG) Rickettsia species (OmpA gene). Of the 24 IFA-positive samples, 5 (21%) were positive for OmpA and 9 (38%) for citrate synthase. In the IFA-negative group (n = 86), OmpA and citrate synthase were positive in 23 (27%) and 27 (31%), respectively. These results showed that the 2 qPCR protocols were about twice as sensitive as the IFA test alone (93% concordance). In conclusion, qPCR is a sensitive method for the diagnosis of fatal BSF cases and should be considered for routine surveillance of AFIHS in places like Brazil, where spotted fever-related lethality is high and other endemic diseases like dengue and leptospirosis can mislead diagnosis. (C) 2012 Elsevier GmbH. All rights reserved.

Morphological records of oocyte maturation in the parthenogenetic tick Amblyomma rotundatum Koch, 1844 (Acari: Ixodidae)

Oocyte maturation in the thelytokous parthenogenetic tick Amblyomma rotundatum was examined for the first time using light and scanning electron microscopy. The panoistic ovary lacks nurse and follicular cells and is a single continuous tubular structure forming a lumen delimited by the ovarian wall. Oocytes of tick species are usually classified according to cytoplasm appearance, the presence of germinal vesicle, the presence of yolk granules, and the chorion. However, for this species, we also use oocyte size as an auxiliary tool since most oocytes were in stages I-Ill and were histologically very similar. Oocytes were classified into five development stages, and specific characteristics were observed: mature oocytes with thin chorion, pedicel cells arranged forming an epithelium with two Or more oocytes attached by the same structure, and a large number of oocytes in the process of reabsorption. (C) 2011 Elsevier GmbH. All rights reserved.

Surveillance using serological and molecular methods for the detection of infectious agents in captive Brazilian neotropic and exotic felids

The aim of the current study was to investigate the exposure of captive wild felids to various infectious pathogens using serological and molecular methods. One hundred and fifty-nine neotropic felids and 51 exotic felids from 28 captive settings in Brazil were tested. While antibodies against Feline parvovirus and Feline coronavirus (FCoV), Feline calicivirus and Bartonella spp. were frequently detected by serologic tests, antibodies against Felid herpesvirus 1 or infection with hemotropic mycoplasmas were less prevalent. Serologic evidence of exposure to Ehrlichia spp., Feline immunodeficiency virus, and Feline leukemia virus (FeLV) was detected rarely, and infections with FeLV, Ehrlichia spp., and Cytauxzoon spp. were found infrequently. The detected Bartonella sequence was molecularly similar to B. koehlerae and B. henselae; for Cytauxzoon, the sequence resembled those from domestic cats. No Anaplasma phagocytophilum and Theileria spp. infections were detected. The positive test results varied significantly among different facilities and species. Additionally, FCoV seropositivity was more prevalent in captivity than in free-ranging populations. Results suggest that testing is appropriate prior to relocation of felids.

Natural infection of triatomines (Hemiptera: Reduviidae) by trypanosomatids in two different environments in the municipality of Ouro Preto do Oeste, State of Rondonia, Brazil

Introduction: This study analyzed the occurrence and the contamination of triatomines by trypanosomatids in Orbignya speciosa (babassu) specimens in the State of Rondonia, Brazil, in two different environments (pasture and woods). Methods: Capture of triatomines on babassus and microscopic search for trypanosomatids in their digestive tube were carried out. Results: Four hundred ninety-four (494) specimens were captured (Rhodnius prolixus and R.robustus), of which 35.6% of the triatomines were positive for trypanosomatids. Conclusions: The high index of natural infection along with the abundance of triatomines points out to the necessity to create an epidemiological surveillance system to monitor vector-borne transmission and deepen the studies on the ecology of such vectors in the Amazon.

Natural infection of triatomines (Hemiptera: Reduviidae) by trypanosomatids in two different environments in the municipality of Ouro Preto do Oeste, State of Rondônia, Brazil

INTRODUCTION: This study analyzed the occurrence and the contamination of triatomines by trypanosomatids in Orbignya speciosa (babassu) specimens in the State of Rondônia, Brazil, in two different environments (pasture and woods). METHODS: Capture of triatomines on babassus and microscopic search for trypanosomatids in their digestive tube were carried out. RESULTS: Four hundred ninety-four (494) specimens were captured (Rhodnius prolixus and R.robustus), of which 35.6% of the triatomines were positive for trypanosomatids. CONCLUSIONS: The high index of natural infection along with the abundance of triatomines points out to the necessity to create an epidemiological surveillance system to monitor vector-borne transmission and deepen the studies on the ecology of such vectors in the Amazon.

A stochastic spatially structured epidemic model with diffusive processes

We developed a stochastic lattice model to describe the vector-borne disease (like yellow fever or dengue). The model is spatially structured and its dynamical rules take into account the diffusion of vectors. We consider a bipartite lattice, forming a sub-lattice of human and another occupied by mosquitoes. At each site of lattice we associate a stochastic variable that describes the occupation and the health state of a single individual (mosquito or human). The process of disease transmission in the human population follows a similar dynamic of the Susceptible-Infected-Recovered model (SIR), while the disease transmission in the mosquito population has an analogous dynamic of the Susceptible-Infected-Susceptible model (SIS) with mosquitos diffusion. The occurrence of an epidemic is directly related to the conditional probability of occurrence of infected mosquitoes (human) in the presence of susceptible human (mosquitoes) on neighborhood. The probability of diffusion of mosquitoes can facilitate the formation of pairs Susceptible-Infected enabling an increase in the size of the epidemic. Using an asynchronous dynamic update, we study the disease transmission in a population initially formed by susceptible individuals due to the introduction of a single mosquito (human) infected. We find that this model exhibits a continuous phase transition related to the existence or non-existence of an epidemic. By means of mean field approximations and Monte Carlo simulations we investigate the epidemic threshold and the phase diagram in terms of the diffusion probability and the infection probability.

Proteome of Rhipicephalus sanguineus tick saliva induced by the secretagogues pilocarpine and dopamine

One dimensional gel electrophoresis was used to separate proteins from the saliva of Rhipicephalus sanguineus female ticks fed on rabbits. Gel slices were subjected to tryptic digestion and analyzed by reversed-phase HPLC followed by MS/MS analysis. The data were compared to a database of salivary proteins of the same tick and to the predicted proteins of the host. Saliva was obtained by either pilocarpine or dopamine stimulation of partially fed ticks. Electrophoretic separations of both yielded products that were identified by mass spectrometry, although the pilocarpine-derived sample was of much better quality. The majority of identified proteins were of rabbit origin, indicating the recycling of the host proteins in the tick saliva, including hemoglobin, albumin, haptoglobin, transferring, and a plasma serpin. The few proteins found that were previously associated with parasitism and blood feeding include 2 glycine-rich, cement-like proteins, 2 lipocalins, and a thyropin protease inhibitor. Among other of the 19 tick proteins identified, albeit with undefined roles, were SPARC and cyclophilin A. This catalog provides a resource that can be mined for secreted molecules that play a role in tick–host interactions.

Reassessment of the taxonomic status of Amblyomma cajennense (Fabricius, 1787) with the description of three new species, Amblyomma tonelliae n. sp., Amblyomma interandinum n. sp. and Amblyomma patinoi n. sp., and reinstatement of Amblyomma mixtum Koch, 1844, and Amblyomma sculptum Berlese, 1888 (Ixodida: Ixodidae)

A reassessment of the taxonomic status of Amblyomma cajennense based on the morphological analyses of ticks from the whole distribution area of the species resulted in the redescription of A. cajennense, the validation of 2 species which had been reduced to synonymy in the past, Amblyomma mixtum and Amblyomma sculptum, and the description and definition of 3 new species, Amblyomma tonelliae n. sp., Amblyomma interandinum n. sp., and Amblyomma patinoi n. sp. This study provides descriptions and redescriptions, scanning electron microscopic and stereomicroscopic images, updated synonymies, information on geographical distributions, and host associations for each of the 6 species. Amblyomma cajennense s.s. is found in the Amazonian region of South America, A. interandinum is reported from the northern part of the Inter-Andean valley of Peru, A. mixtum is present from Texas (U.S.A.) to western Ecuador, A. patinoi occurs in the Eastern Cordillera of Colombia, A. tonelliae is associated with the dry areas of the Chaco region which spans from central-northern Argentina to Bolivia and Paraguay, whereas A. sculptum is distributed from the humid areas of northern Argentina, to the contiguous regions of Bolivia and Paraguay and the coastal and central-western states of Brazil.

Patogeni e zecche vettrici: studio epidemiologico in parchi pubblici dell'Emilia Romagna

Le malattie trasmesse da zecche sono un importante problema sia per la salute animale che per quella umana e negli ultimi decenni hanno aumentato notevolmente la loro diffusione, in seguito ai cambiamenti climatici, che hanno permesso la distribuzione delle zecche in aree prima non interessate. Per tale motivo si è deciso di effettuare un’indagine sulla diffusione delle zecche e sui patogeni da loro trasmessi, mediante campionamenti sia a livello ambientale, sia su animali e umani infestati in quattro siti di tre parchi dell’Emilia Romagna, dove non risultavano precedenti segnalazioni, nelle province di Bologna e Ravenna, da Aprile a Ottobre 2010. In totale sono state raccolte 8212 zecche. Dall’ambiente sono state campionate 6734 larve, 1344 ninfe, 61 adulti; dagli animali e da persone sono stati raccolti 68 adulti e 5 ninfe appartenenti a diverse specie di Ixodidae. Sono state condotte analisi sull’abbondanza delle zecche nelle diverse aree di raccolta, in funzione del periodo di campionamento, della temperatura e dell’umidità relativa misurata a 5 cm dal suolo al momento del campionamento e della vegetazione. Su tutti gli individui adulti e su pool di ninfe e di larve, per un totale di 393 campioni, sono state condotte analisi di tipo molecolare per la ricerca di piroplasmi, Anaplasma phagocytophilum e Borrelia burgdorferi s.l. Attraverso la PCR e il sequenziamento, è emerso che il 7,6% dei campioni era positivo per piroplasmi, tra i quali è stata riscontrata anche la presenza delle specie zoonosiche Babesia EU1 e B. divergens. La real-time PCR eseguita solo sui campioni costituiti da ninfe e adulti ha evidenziato una prevalenza del 9,2% per A. phagocytophilum e del 21,6% per B. burgdorferi s.l. Su questi patogeni sono state quindi condotte analisi di tipo filogenetico. In alcuni campioni sono state riscontrate coinfezioni con combinazioni di due patogeni contemporaneamente.

Evaluation of Microbial Contamination in Bivalve Mollusks: Epidemiology and Diagnosis

Shellfish are filter-feeding organisms that can accumulate many bacteria and viruses. Considering that depuration procedures are not effective in removal of certain microorganisms, shellfish-borne diseases are frequent in many parts of the world, and their control must rely primarily on investigation of prevalence of human pathogens in shellfish and water environment. However, the diffusion of enteric viruses and Vibrio bacteria is not known in many geographical areas, for example in Sardinia, Italy. A survey aimed at investigating the prevalence of Norovirus (NoV), hepatitis A virus (HAV), V. parahaemolyticus, V. cholerae and V. vulnificus was carried out, analyzing both local and imported purified, non-purified and retail shellfish from North Italy and Sardinia. Shellfish from both areas were found contaminated by NoVs, HAV and Vibrio, including retail and purified animals. Molecular analysis evidenced different NoV genogroups and genotypes, including bovine NoVs, as well as pathogenic Vibrio strains, underlining the risk for shellfish consumers. However, also other approaches are needed to control the diffusion of shellfish-borne diseases. It was originally thought that enteric viruses are passively accumulated by shellfish. Recently, it was proven that NoVs bind to specific carbohydrate ligands in oysters, and various NoV strains are characterized by a different bioaccumulation pattern. To deepen the knowledge on this argument, a study was carried out, analyzing bioaccumulation of up to 8 different NoV strains in four different species of shellfish. Different bioaccumulation patterns were observed for each shellfish species and NoV strain used, potentially important in setting up effective shellfish purification protocols. Finally, a novel study of evaluation of viral contamination in shellfish from the French Atlantic coast was carried out following the passage of Xynthia tempest over Western Europe which caused massive destruction. Different enteric viruses were found over a one month period, evidencing the potential of these events of contaminating shellfish.

Gene expression analysis in ‘Candidatus Phytoplasma mali’-resistant and -susceptible Malus genotypes

Apple proliferation (AP) disease is the most important graft-transmissible and vector-borne disease of apple in Europe. ‘Candidatus Phytoplasma mali’ (Ca. P. mali) is the causal agent of AP. Apple (Malus x domestica) and other Malus species are the only known woody hosts. In European apple orchards, the cultivars are mainly grafted on one rootstock, M. x domestica cv. M9. M9 like all other M. x domestica cultivars is susceptible to ‘Ca. P. mali’. Resistance to AP was found in the wild genotype Malus sieboldii (MS) and in MS-derived hybrids but they were characterised by poor agronomic value. The breeding of a new rootstock carrying the resistant and the agronomic traits was the major aim of a project of which this work is a part. The objective was to shed light into the unknown resistance mechanism. The plant-phytoplasma interaction was studied by analysing differences between the ‘Ca. P. mali’-resistant and -susceptible genotypes related to constitutively expressed genes or to induced genes during infection. The cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technique was employed in both approaches. Differences related to constitutively expressed genes were identified between two ‘Ca. P. mali’-resistant hybrid genotypes (4551 and H0909) and the ‘Ca. P. mali’-susceptible M9. 232 cDNA-AFLP bands present in the two resistant genotypes but absent in the susceptible one were isolated but several different products associated to each band were found. Therefore, two different macroarray hybridisation experiments were performed with the cDNA-AFLP fragments yielding 40 sequences encoding for genes of unknown function or a wide array of functions including plant defence. In the second approach, individuation and analysis of the induced genes was carried out exploiting an in vitro system in which healthy and ‘Ca. P. mali’-infected micropropagated plants were maintained under controlled conditions. Infection trials using in vitro grafting of ‘Ca. P. mali’ showed that the resistance phenotype could be reproduced in this system. In addition, ex vitro plants were generated as an independent control of the genes differentially expressed in the in vitro plants. The cDNA-AFLP analysis in in vitro plants yielded 63 bands characterised by over-expression in the infected state of both the H0909 and MS genotypes. The major part (37 %) of the associated sequences showed homology with products of unknown function. The other genes were involved in plant defence, energy transport/oxidative stress response, protein metabolism and cellular growth. Real-time qPCR analysis was employed to validate the differential expression of the genes individuated in the cDNA-AFLP analysis. Since no internal controls were available for the study of the gene expression in Malus, an analysis on housekeeping genes was performed. The most stably expressed genes were the elongation factor-1 α (EF1) and the eukaryotic translation initiation factor 4-A (eIF4A). Twelve out of 20 genes investigated through qPCR were significantly differentially expressed in at least one genotype either in in vitro plants or in ex vitro plants. Overall, about 20% of the genes confirmed their cDNA-AFLP expression pattern in M. sieboldii or H0909. On the contrary, 30 % of the genes showed down-regulation or were not differentially expressed. For the remaining 50 % of the genes a contrasting behaviour was observed. The qPCR data could be interpreted as follows: the phytoplasma infection unbalance photosynthetic activity and photorespiration down-regulating genes involved in photosynthesis and in the electron transfer chain. As result, and in contrast to M. x domestica genotypes, an up-regulation of genes of the general response against pathogens was found in MS. These genes involved the pathway of H2O2 and the production of secondary metabolites leading to the hypothesis that a response based on the accumulation of H2O2 in MS would be at the base of its resistance. This resembles a phenomenon known as “recovery” where the spontaneous remission of the symptoms is observed in old susceptible plants but occurring in a stochastic way while the resistance in MS is an inducible but stable feature. As additional product of this work three cDNA-AFLP-derived markers were developed which showed independent distribution among the seedlings of two breeding progenies and were associated to a genomic region characteristic of MS. These markers will contribute to the development of molecular markers for the resistance as well as to map the resistance on the Malus genome.

Questing Dermacentor reticulatus harbouring Babesia canis DNA associated with outbreaks of canine babesiosis in the Swiss Midlands

In 2011 and 2012, outbreaks of clinical canine babesiosis were observed in 2 areas of the Swiss Midlands that had no history of this disease so far. In one area, cases of canine babesiosis occurred over 2 consecutive tick seasons. The outbreaks involved 29 dogs, 4 of which died. All dogs were infected with large Babesia sp. as diagnosed in Giemsa-stained blood smears and/or PCR. These were identified as B. canis (formerly known as B. canis canis) by subsequent partial sequencing of the 18S rRNA gene of Babesia sp. Interestingly, the sequence indicated either a genotype with heterogeneity in the ssrRNA gene copies or double infection with different B. canis isolates. None of the dogs had a recent travel history, but one had frequently travelled to Hungary and had suffered twice from clinical babesiosis 18 and 24 months prior to the outbreak in autumn 2011. Retrospective sequencing of a stored blood DNA sample of this dog revealed B. canis, with an identical sequence to the Babesia involved in the outbreaks. For the first time in Switzerland, the partial 18S rRNA gene of B. canis could be amplified from DNA isolated from 19 out of 23 adult Dermacentor reticulatus ticks flagged in the same area. The sequence was identical to that found in the dogs. Furthermore, one affected dog carried a female D. reticulatus tick harbouring B. canis DNA. Our findings illustrate that, under favourable biogeographic and climatic conditions, the life-cycle of B. canis can relatively rapidly establish itself in previously non-endemic areas. Canine babesiosis should therefore always be a differential diagnosis when dogs with typical clinical signs are presented, regardless of known endemic areas.

A century of bovine besnoitiosis: an unknown disease re-emerging in Europe.

Bovine besnoitiosis, which is caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti, is a chronic and debilitating vector-borne disease characterized by both cutaneous and systemic manifestations. In Europe, this parasitic disease appeared in a few restricted areas in France and Portugal since the first recorded cases in the beginning of the 20th century. However, at present, the disease is considered to be re-emerging by the European Food Safety Authority due to an increased number of cases and the geographic expansion of besnoitiosis into cattle herds in several European countries. In this review, we will provide an update of the epidemiology and impact of B. besnoiti infection. Strategies to control this parasitic disease will also be discussed.