Luís Madeira de Carvalho

Survey of infectious and parasitic diseases in stray cats at the Lisbon Metropolitan Area, Portugal

A survey of infectious and parasitic diseases of stray cats was carried out using biological samples collected from animals captured during a catch-neuter-release programme in four counties of the Lisbon Metropolitan Area. The main objective was to investigate the potential threat of stray cats for animal and public health. Samples of blood, stool, hair and auricular swabs were collected from 231 cats in 27 colonies. Anti-Toxoplasma gondii antibodies were detected in 47/194 samples (24.2%); anti-Leishmania infantum antibodies in 1/180 cats (0.6%); intestinal parasites in 23/74 samples (Toxocara cati, Isospora felis, Ancylostoma tubaeforme, Dipylidium caninum, Uncinaria stenocephala, Toxascaris leonina) and Otodectes cynotis in 4/182 cats (2.2%); dermatophyte fungi were isolated in 40/136 samples (29.4%); feline immunodeficiency virus antibodies were detected in 23/226 samples (10.2%); feline leukaemia virus antigen in 14/198 samples (7.1%); and feline coronavirus RNA in 9/127 samples (7.1%). Our results revealed that zoonotic agents, namely dermatophyte fungi and Toxocara cati were present in stray cat colonies in the investigated counties. Overall the low frequency of major pathogens suggests a balanced relationship between host and agents.

Prevalence of Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi sensu lato, Anaplasma spp. and Leishmania infantum in apparently healthy and CVBD-suspect dogs in Portugal - a national serological study

BackgroundCanine vector-borne diseases (CVBDs) are caused by a wide range of pathogens transmitted to dogs by arthropods including ticks and insects. Many CVBD-agents are of zoonotic concern, with dogs potentially serving as reservoirs and sentinels for human infections. The present study aimed at assessing the seroprevalence of infection with or exposure to Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi sensu lato, Anaplasma spp. and Leishmania infantum in dogs in Portugal.MethodsBased on 120 veterinary medical centres from all the regions of mainland and insular Portugal, 557 apparently healthy and 628 CVBD-suspect dogs were sampled. Serum, plasma or whole blood was tested for qualitative detection of D. immitis antigen and antibodies to E. canis, B. burgdorferi s. l., Anaplasma spp. and L. infantum with two commercial in-clinic enzyme-linked immunosorbent assay kits. Odds ratios (OR) were calculated by logistic regression analysis to identify independent risk factors of exposure to the vector-borne agents.ResultsTotal positivity levels to D. immitis, E. canis, B. burgdorferi, Anaplasma spp., L. infantum, one or more agents and mixed agents were 3.6%, 4.1%, 0.2%, 4.5%, 4.3%, 14.0% and 2.0% in the healthy group, and 8.9%, 16.4%, 0.5%, 9.2%, 25.2%, 46.3% and 11.6% in the clinically suspect group, respectively. Non-use of ectoparasiticides was a risk factor for positivity to one or more agents both in the apparently healthy (OR = 2.1) and CVBD-suspect (OR = 1.5) dogs. Seropositivity to L. infantum (OR = 7.6), E. canis (OR = 4.1) and D. immitis (OR = 2.4) were identified as risk factors for the presence of clinical signs compatible with CVBDs. Positivity to mixed agents was not found to be a risk factor for disease.ConclusionsDogs in Portugal are at risk of becoming infected with vector-borne pathogens, some of which are of zoonotic concern. CVBDs should be considered by practitioners and prophylactic measures must be put in place to protect dogs and limit the risk of transmission of vector-borne agents to humans. This study is expected to give veterinary and public health authorities an increased awareness about CVBDs in Portugal and to serve as a reference for future investigations and control actions.

Mutation scanning-based analysis of Theileria orientalis populations in cattle following an outbreak

Bovine theileriosis is a tick‐borne disease caused by one or more hemoprotozoan parasites of the genus Theileria. In the past, Theileria infection in cattle in Australia was largely asymptomatic and recognized to be associated with Theileria buffeli. However, outbreaks of theileriosis have occurred in beef and dairy cattle in subtropical climatic regions (New South Wales) of Australia. There is also one published report of a recent theileriosis outbreak in a beef farm near Seymour in the southeastern state of Victoria. In order to gain an improved insight into the genetic composition of Theileria populations following this outbreak, we undertook herein an integrated PCR‐coupled mutation scanning‐sequencing‐phylogenetic analysis of sequence variation in part of the major piroplasm surface protein (MPSP) gene within and among samples from cattle involved in the outbreak. Theileria DNA was detected in 89.4% of 94 cattle in the Seymour farm; the genetic analysis showed that the ikeda and chitose genotypes representing the Theileria orientalis complex were detected in 75 and 4.8% of 84 infected cattle, respectively, and that mixed populations of these two genotypes were found in 20.2% of infected cattle. Given unpublished reports of a significant increase in the number of outbreaks in Victoria, future investigations should focus sharply on elucidating the epidemiology of Theileria to subvert the economic impact on the cattle industry in this state. Although used here to explore genetic variation within the T. orientalis complex in Australia, a mutation scanning‐based approach has broad applicability to other species of Theileria in other countries.

Taeniid species of the Iberian wolf (Canis lupus signatus) in Portugal with special focus on Echinococcus spp.

Graphical abstract Highlights ► This is the first study on the taeniid species infecting the Portuguese population of Iberian wolf. ► Taeniid species detected are strongly related to the feeding habits of this top carnivore. ► First report of Taenia polyacantha and Echinococcus intermedius in the Iberian wolf. ► A genuine wild cycle for E. intermedius might persist between wolves and wild boars in this region.

Molecular detection of Rangelia vitalii in domestic dogs from Uruguay.

The piroplasm Rangelia vitalii is the etiological agent of canine rangeliosis, a severe disease affecting domestic dogs in South America. Two domestic dogs from two different Departments (Salto and Treinta y Tres) of Uruguay presented with clinical signs such as apathy, anorexia, pale mucous membranes, jaundice, and hemorrhagic manifestations, suggestive of a canine vector-borne disease. Molecular analysis, based on PCR and DNA sequencing of portions of the 18S rRNA gene, revealed that both dogs were infected by R. vitalii. Two consensus sequences, one from Salto and one from Treinta y Tres, differed from each other by only 1 nucleotide (99.8% similarity) and were 99.8-100% identical to corresponding sequences of R. vitalii from Brazil and Argentina available in GenBank. Through phylogenetic analysis inferred by the 18S rRNA gene, the two Uruguayan sequences of R. vitalii were aligned with the corresponding sequences from 7 other R. vitalii sequences available in GenBank (5 from Brazil and, 2 from Argentina) under high bootstrap support. The two dogs of the present study were negative for Ehrlichia canis according to the E. canis-specific real-time PCR assay. Our findings not only confirm the occurrence of R. vitalii in Uruguay but also provide the southernmost record of this re-emerging agent. The only previous report of R. vitalii in Uruguay dated from 1976, a period when molecular analyses were not available. We provide the first molecular detection of R. vitalii in Uruguay. Currently, canine rangeliosis is confirmed to occur in Brazil, Argentina, and Uruguay.

Prevalence and seasonal variations of canine dirofilariosis in Portugal.

Dirofilariosis is a severe vector-borne emergent disease that is spreading worldwide and becoming a serious threat to human and veterinary public health. Portugal, a Mediterranean country, has favorable climate conditions for mosquito development and survival. At present, accurate data on the prevalence and epidemiological pattern of dirofilariosis in Portugal is scarce and outdated. To study these trends, a project was developed to assess the current prevalence of Dirofilaria immitis infection in shelter dogs as well as the prevalence of Dirofilaria species present in three coastal regions in central Portugal: Coimbra, Santarém, and Setúbal. Blood samples were collected from 696 shelter dogs during three consecutive years: 2011, 2012, and 2013. A rapid immunomigration technique was performed to detect female D. immitis antigens. Concurrently, to detect and identify circulating microfilariae, a modified Knotts technique and acid phosphatase histochemical staining were also performed. Of the 696 dogs sampled, 105 were positive for D. immitis, with an overall prevalence of 15.1%. Forty of the 105 dogs were antigen negative but were positive for D. immitis microfilariae. Three animals were co-infected with D. immitis and Acanthocheilonema dracunculoides, and there was also one dog infected only with A. dracunculoides, all confirmed by polymerase chain reaction. The highest prevalence of canine heartworm during the 3-y period was in Setúbal (24.8%), followed by Coimbra (13.8%), and Santarém (13.2%), with significant inter-district differences. Our results demonstrate a higher prevalence of dirofilariosis compared with findings of previous studies and show an increasing rate of infection in the southern areas of Portugal attributed, at least in part, to bioclimatic and ecological factors. The present study updates the epidemiological situation and correlates the risk of dirofilariosis transmission within each region. These findings are highly relevant to both human and veterinary public health, contributing to the general awareness of pet owners and veterinarian practitioners and reinforcing the need for effective control measures against vectors and preventive therapy in companion animals.

Feline thelaziosis caused by Thelazia callipaeda in Portugal

Thelazia callipaeda (Spirurida, Thelaziidae) is a nematode that lives in the conjunctival sac of domestic and wild carnivores, rabbits and humans causing mild to severe symptoms (e.g., conjunctivitis, lacrimation, epiphora, blepharospasm, keratitis and even corneal ulceration) in infected animals. This report describes an autochthonous case of thelaziosis in a cat from the central region of Portugal, representing the most occidental record of thelaziosis in Europe. Adult nematodes recovered from alive animal were morphological identified as T. callipaeda. A portion of the mitochondrial cytochrome c oxidase subunit 1 gene (cox 1) from nematode specimens was amplified by PCR. Cox1 sequences of all specimens were identical to T. callipaeda haplotype 1. Additionally to these findings, a recent description of thelaziosis in the northern region of Portugal suggests that T. callipaeda has successfully established in Portugal.

Lungworms and gastrointestinal parasites of domestic cats: a European perspective

With the exception of Aelurostrongylus abstrusus, feline lungworms have been poorly studied. Information on their distribution is patchy and mostly limited to case reports. In this study, the occurrence of feline lungworms and co-infecting gastrointestinal parasites has been investigated in 12 European countries (i.e. Austria, Belgium, Bulgaria, France, Greece, Hungary, Italy, Portugal, Romania, Spain, Switzerland and the United Kingdom). An average of 10 domestic cats, with regular outdoor access, was sampled each month for 12months, and freshly passed faeces were collected. Stools were processed using a McMaster assay and a quantitative Baermann-Wetzel method. Animals positive for lungworms and/or gastrointestinal parasites were treated with a formulation containing fipronil, (S)-methoprene, eprinomectin, and praziquantel (Broadline®, Merial), and re-sampled 28days post-treatment. The association between lungworm infection and risk factors was analysed using statistical medians/means and the efficacy of the treatment against each lungworm species was assessed. Of 1990 cats sampled, 613 (30.8%) were positive for at least one parasite, while 210 (10.6%) were infected by lungworms. The prevalence of lungworm infection varied between the sampled sites, with the highest recorded in Bulgaria (35.8%) and the lowest in Switzerland (0.8%). None of the cats from Austria or the United Kingdom were infected by lungworms. Aelurostrongylus abstrusus was the species most frequently detected (78.1%), followed by Troglostrongylus brevior (19.5%), Eucoleus aerophilus (14.8%) and Oslerus rostratus (3.8%). The overall efficacy of the treatment was 99% for A. abstrusus and 100% for T. brevior, O. rostratus and E. aerophilus. Data presented provide a comprehensive account of the diagnosis, epidemiology and treatment of feline lungworms in Europe, as well as of the occurrence of co-infections by gastrointestinal parasites.

Helminth infections and gut microbiota – a feline perspective

BackgroundInvestigations of the relationships between the gut microbiota and gastrointestinal parasitic nematodes are attracting growing interest by the scientific community, driven by the need to better understand the contribution of parasite-associated changes in the composition of the gut flora to both host malnutrition and immune modulation. These studies have however been carried out mainly in humans and experimental animals, while knowledge of the make-up of the gut commensal flora in presence or absence of infection by parasitic nematodes in domestic animals is limited. In this study, we investigate the qualitative and quantitative impact that infections by a widespread parasite of cats (i.e. Toxocara cati) exert on the gut microbiota of feline hosts.MethodsThe faecal microbiota of cats with patent infection by T. cati (= Tc+), as well as that of negative controls (= Tc-) was examined via high-throughput sequencing of the V3-V4 hypervariable region of the bacterial 16S rRNA gene, followed by bioinformatics and biostatistical analyses of sequence data.ResultsA total of 2,325,366 useable high-quality sequences were generated from the faecal samples analysed in this study and subjected to further bioinformatics analyses, which led to the identification of 128 OTUs and nine bacterial phyla, respectively. The phylum Firmicutes was predominant in all samples analysed (mean of 53.0%), followed by the phyla Proteobacteria (13.8%), Actinobacteria (13.7%) and Bacteroidetes (10.1%). Among others, bacteria of the order Lactobacillales, the family Enterococcaceae and genera Enterococcus and Dorea showed a trend towards increased abundance in Tc+  compared with Tc- samples, while no significant differences in OTU richness and diversity were recorded between Tc+ and Tc- samples (P = 0.485 and P = 0.581, respectively). However, Canonical Correlation and Redundancy Analyses were able to separate samples by infection status (P = 0.030 and P = 0.015, respectively), which suggests a correlation between the latter and the composition of the feline faecal microbiota.ConclusionsIn spite of the relatively small number of samples analysed, subtle differences in the composition of the gut microbiota of Tc+ vs Tc- cats could be identified, some of which in accordance with current data from humans and laboratory animal hosts. Nevertheless, the findings from this study contribute valuable knowledge to the yet little explored area of parasite-microbiota interactions in domestic animals.

Molecular detection of vector-borne pathogens in dogs and cats from Qatar

BackgroundVector-borne diseases (VBDs) have been increasingly reported in dogs and cats worldwide. However, no data are currently available regarding canine and feline VBDs in Qatar and limited information is available from other Persian Gulf countries.MethodsBlood samples from 98 client-owned animals (i.e. 64 dogs and 34 cats) living in Doha (Qatar) were collected and the presence of genomic DNA of Anaplasma spp., Babesia spp., Dirofilaria spp., Ehrlichia spp., Hepatozoon spp., Mycoplasma spp. and Rickettsia spp. was assessed by polymerase chain reaction (PCR), real time-PCR (rt-PCR) and sequence analysis.ResultsOf the 64 dogs, 12 (18.8%) were infected with at least one pathogen (i.e. 7.8% with Mycoplasma spp., 4.7% with Babesia vogeli, 3.1% with Ehrlichia canis, and 1.6% with Anaplasma platys, Babesia gibsoni and Hepatozoon canis, each). One of the 12 dogs was co-infected with B. vogeli and E. canis. Of the 34 cats, seven (20.6%) animals were infected with at least one pathogen (i.e. 5.9% were positive for Mycoplasma spp., and 2.9% for Babesia felis, B. vogeli, E. canis, “Candidatus Mycoplasma haemominutum” and Mycoplasma haemofelis, each). No dogs or cats were positive for Dirofilaria spp. or Rickettsia spp.ConclusionsAlthough the sample sizes of dogs and cats herein analysed was moderately small, data from this study report the occurrence of A. platys, B. vogeli, B. gibsoni, E. canis, H. canis and Mycoplasma spp. in domestic dogs and of B. felis, B. vogeli, “Candidatus M. haemominutum”, E. canis and M. haemofelis in domestic cats from Qatar. Further investigations along with prophylactic measures are strongly recommended in order to reduce the risk of dogs and cats acquiring VBDs in Qatar.