Genetic and virulence characterization of Toxoplasma gondii strains isolated from pigeons in Lisbon region

habitat with cats and humans, bands are observed in recreational areas such as urban parks, playgrounds and parks. The interaction between cats, birds and human population is quite evident favoring the fecal-oral transmission of T. gondii between the definitive host and intermediate hosts, in the urban cycle of the parasite. The results of the inoculation in vivo of the brain homogenates showed pigeon isolation rates (58.5%) significantly higher when compared with previous studies, including the preliminary study in 2006 that the isolation rate in mice was 39,1% (9/23) (Waap 2008) and another that was not achieved any isolation in mouse (Godoi 2010). The genotypic analysis revealed a majority of strains of type II, which is consistent with what has been described in Portugal, the rest of Europe and the USA (Ajzenberg 2005, Fazaeli 2000, Honoré 2000, Howe 1997, Waap 2008) . We also isolated strains of type III and type I. The identification of type III strains in animals have been reported by other authors, but the type I have been rarely found in animals has not been previously described in Portugal except in a preliminary study of our team at the 2008 (Waap 2008). The type I strains are usually associated with high virulence in laboratory mice, leading to death within days. This strain was identified by molecular biology and has not been isolated in vivo. The difficulty in isolation of strain may be related to the small number of cysts of the type I strains can develop, these type strains are considered low cystogenic. Genetic characterization of strains of T. gondii is far from its terminus, more sequences of different genes should be studied to help the understanding of the molecular epidemiology and genetic characterization of T. gondii, a relevant parasite for which these data are lacking. The combination of data from humans and animals, through the use of high resolution genetic characterization should improve our perceptive of T. gondii, which will be ultimately beneficial for the control of T. gondii transmission

Isolamento de Toxoplasma gondii a partir de cérebro e músculo de gatos serologicamente positivos utilizando culturas celulares

O isolamento de Toxoplasma gondii é imprescindível quer para efeitos de diagnóstico definitivo da infecção, quer para estudos visando a genotipagem de estirpes circulantes, tanto nos animais como no ser humano. O bioensaio é presentemente considerado o método de eleição para isolamento de T. gondii de amostras biológicas, no entanto, para além de eticamente questionável, a inoculação em ratinho é dispendiosa e laboriosa. O ensaio in vitro utilizando culturas celulares é frequentemente mencionado como alternativa, contudo o investimento nesta área tem sido diminuto, sendo raros os trabalhos que referem o recurso a este método para isolamento de T. gondii a partir de tecidos animais. Este estudo teve como objectivo testar diferentes métodos de isolamento de T. gondii em culturas de células. Para a preparação dos inóculos, colhemos amostras de tecido cerebral e muscular (coração e membros) de 16 gatos serologicamente positivos, durante a necrópsia. O tecido cerebral foi homogeneizado com agulha e seringa em meio de cultura e o tecido muscular digerido numa solução de ácido clorídrico e pepsina. A inoculação dos homogeneizados de cérebro e músculo em culturas de células Vero resultou, respectivamente, numa taxa de isolamento de 37,5% (6/16) e 62,5% (10/16). A visualização microscópica de taquizoítos nas culturas celulares foi possível 5-14 dias pós-inoculação, utilizando homogeneizados de cérebro e 7-32 dias pós-inoculação, utilizando homogeneizados de tecido muscular. Todos os isolados foram confirmados por n-PCR visando a região ITS1 do rDNA de T. gondii

Isolation of Besnoitia besnoiti from infected cattle in Portugal

Abstract Besnoitia besnoiti, an obligate intracellular protozoan parasite belonging to the phylum apicomplexa, is the causative agent of bovine besnoitiosis. Besnoitiosis is responsible for significant losses in the cattle industry of Africa and Mediterranean countries due to the high morbidity rate, abortion and infertility in males. The acute stage of disease is associated with the proliferative forms (tachyzoites) and is characterized by fever, whimpery, general weakness and swelling of the superficial lymph nodes. During the following chronic stage, a huge number of cysts are formed mainly in the subcutaneous tissues. This process is non-reversible, and chronic besnoitiosis is characterized by hyper-sclerodermia, hyperkeratosis, alopecia and, in bulls, atrophy, sclerosis and focal necrosis that cause irreversible lesions in the testis. In this paper we report on the identification of large cysts in the skin of a cow and a bull in Portugal, which presented loss of hair and enlargement and pachydermis all over the body. The observation of a two-layered cyst wall within the host cell, the encapsulation of the host cell by a large outer cyst wall, and the subcutaneous localization of the cysts within the host, were characteristic for B. besnoiti. The parasites were isolated from the infected animals and successfully propagated in Vero cells without prior passages in laboratory animals. Morphological characterization of B. besnoiti tachyzoites and the amplification of the 149 bp segment from the internal transcribed spacer 1 (ITS1), aided with specific primers, confirmed the identification of B. besnoiti. Keywords: Besnoitia besnoiti; Bovine besnoitiosis; Portugal; Cyst; In vitro cell cultur

ÁCAROS TROMBICULÍDEOS: REVISÃO DE UMA PARASITOSE NEGLIGENCIADA EM ANIMAIS DE COMPANHIA

A trombiculose é uma parasitose causada por ácaros trombiculídeos (Acari: Trombiculidae) que afeta vertebrados, incluindo cães, gatos e o ser humano. É a fase larvar dos ácaros trombiculídeos que se caracteriza por ser parasita obrigatório e responsável pelo aparecimento de sinais clínicos. Estes incluem prurido, várias lesões cutâneas, podendo, em alguns casos, surgir distúrbios gastrointestinais, neurológicos ou mesmo a morte do animal quando as infestações são massivas. Apesar da sua distribuição mundial, incluindo em Portugal, esta é uma parasitose negligenciada, sobretudo no contexto da medicina veterinária de animais de companhia. Os estudos existentes são escassos, contribuindo para o seu desconhecimento, e a sua verdadeira prevalência nestes animais é pouco conhecida a nível mundial. Apesar do seu papel enquanto vetores de agentes patogénicos não ser claro, alguns estudos já demonstraram a presença de ADN de Anaplasma spp., Bartonella spp., Borrelia spp. e Rickettsia spp., em trombiculídeos. O facto de os ácaros trombiculídeos afetarem várias espécies animais, incluindo os animais de companhia e o ser humano, bem como o seu potencial papel no ciclo de manutenção de vários agentes infeciosos, revela a importância de um maior conhecimento acerca desta parasitose, tanto ao nível da comunidade científica como da comunidade médico veterinária. Assim, esta revisão pretende reunir os dados disponíveis à data quanto à epidemiologia, características clínicas e tratamento desta parasitose, com especial enfoque em Portugal

Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method

Purpose: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. Methods: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. Results: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. Conclusion: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution

A modified agglutination test for the diagnosis of Besnoitia besnoiti infection

Abstract Bovine besnoitiosis is caused by Besnoitia besnoiti, an obligate intracellular apicomplexan parasite. Affected animals present cutaneous and systemic manifestations and the disease may lead to considerable economic losses. Although generally associated to tropical and subtropical areas, bovine besnoitiosis is now considered an emergent disease in Europe, due to the increasing number of new cases and apparent geographical expansion. In this study we evaluated the performance of a modified agglutination test (B-MAT) in the serodiagnosis of bovine besnoitiosis in comparison to the indirect immunofluorescent-antibody test (IFAT). To establish optimal protocol conditions we used bovine sera with a known infection status for B. besnoiti infection. Positive animals (n=36) presented B. besnoiti dermal cysts and anti-B. besnoiti specific antibodies, as determined by the indirect immunofluorescence test (IFAT). Negative animals (n=103) were from non-endemic areas in Portugal and negative by the IFAT. From here, we evaluated the sensitivity and specificity of the B-MAT relative to the IFAT with a panel of sera from herds with history of bovine besnoitiosis in Portugal, Spain and France (n=402), using three serum dilutions (1:80, 1:160, 1:320). Considering the positive cut-off at 1:160 serum dilution, the B-MAT showed an almost perfect test agreement with the IFAT; (κ=0.968; 95% CI: 0.941-0.996) with a relative sensitivity of 97.2% (95% CI: 94.1-100%) and a relative specificity of 99.3% (95% CI: 98.4-100%). As a simple and inexpensive technique the B-MAT represents a valuable tool for the diagnosis and study of the epidemiology of bovine besnoitiosis

Tree file of cephalopods

RAxML Tree used for taxonomic assignment of 16SrRNA sequences of cephalopod

Alignment of 16SRNA cephalopod sequences

Aligned 16SrRNA mitochondrial sequences of the cephalopod specimens collected in the stomach contents of Bulweria bulwerii and reference sequence

Data from: The diet of a nocturnal pelagic predator, the Bulwer’s petrel, across the lunar cycle

The lunar cycle is believed to strongly influence the vertical distribution of many oceanic taxa, with implications for the foraging behaviour of nocturnal marine predators. Most studies to date testing lunar effects on foraging have focused on predator activity at-sea, with some birds and marine mammals demonstrating contrasting behavioural patterns, depending on the lunar-phase. However, to date no study has focused on how the lunar cycle might actually affect predator-prey interactions in the upper layers of the ocean. Here, we tested whether the diet of the predominantly nocturnal pelagic predator, the Bulwer’s petrel (Bulweria bulwerii) would change throughout the lunar cycle, using molecular analysis to augment detection and taxonomic resolution of prey collected from stomach-contents. We found no evidence of dietary shifts in species composition or diversity, with Bulwer’s petrel always consuming a wide range of mesopelagic species. Other co-variables potentially affecting light availability at-sea, such as percentage of cloud cover, did not confound our results. Moreover, many of the species found are thought not to reach the sea-surface. Our findings reveal that nocturnal predators are probably more specialized than previously assumed, irrespective of ambient-light, but also reveal deficiencies in our current understanding of species vertical distribution and predation-dynamics at-sea