Coxiella burnetii in Dogs and Cats from Portugal: Serological and Molecular Analysis.

Dogs and cats are potential sources of infection for some zoonotic diseases such as Q fever, caused by Coxiella burnetii, a multiple host pathogen. Q fever outbreaks in dogs and cats have been related with parturition and abortion events, and ticks have a potential role in the transmission of this pathogen. This study aimed to screen for C. burnetii in dogs and cats, and in ticks collected from infested animals. An observational descriptive study was conducted in Portugal at two time points nine years apart, 2012 and 2021. Sera obtained from dogs and cats (total n = 294) were tested for C. burnetii antibodies using a commercial ELISA adapted for multi-species detection. C. burnetii DNA was screened by qPCR assay targeting IS1111 in uterine samples and in ticks. A decrease in the exposure to C. burnetii was observed in cats from 17.2% (95% CI: 5.8−35.8%) in 2012 to 0.0% in 2021, and in dogs from 12.6% (95% CI: 7.7−19.0%) in 2012 to 1.7% (95% CI: 0.3−9.1%) in 2021 (p < 0.05). Overall, and despite differences in the samples, rural habitat seems to favour the exposure to C. burnetii. The DNA of C. burnetii was not detected in ticks. The low seropositivity observed in 2021 and the absence of C. burnetii DNA in the tested samples, suggest that dogs and cats from Portugal are not often exposed to the pathogen. Nevertheless, the monitoring of C. burnetii infection in companion animals is an important tool to prevent human outbreaks, considering the zoonotic potential for owners and veterinarians contacting infected animals, mainly dogs and cats from rural areas which often come into contact with livestock.

Role of Goats in the Epidemiology of Coxiella burnetii.

Since its first description in the late 1930s, Q fever has raised many questions. Coxiella burnetii, the causative agent, is a zoonotic pathogen affecting a wide range of hosts. This airborne organism leads to an obligate, intracellular lifecycle, during which it multiplies in the mononuclear cells of the immune system and in the trophoblasts of the placenta in pregnant females. Although some issues about C. burnetii and its pathogenesis in animals remain unclear, over the years, some experimental studies on Q fever have been conducted in goats given their excretion pattern. Goats play an important role in the epidemiology and economics of C. burnetii infections, also being the focus of several epidemiological studies. Additionally, variants of the agent implicated in human long-term disease have been found circulating in goats. The purpose of this review is to summarize the latest research on C. burnetii infection and the role played by goats in the transmission of the infection to humans.

Gastrointestinal Parasitism in Miranda Donkeys: Epidemiology and Selective Control of Strongyles Infection in the Northeast of Portugal.

In Portugal, equine parasitism in pasture animals is characterized by high parasitic burden and high helminthic biodiversity; both factors are potentially pathogenic for their hosts. The decrease in the number of donkeys over the last years in Portugal, their importance in rural lowland and mountain ecosystems and pastures and the scarce information regarding their parasitism led to this research, which aimed to evaluate the parasitological status of a Miranda donkey breed population, a native breed mainly located in the northeast of Portugal. This study provides better knowledge of their gastrointestinal parasitism, particularly strongyles, and the assessment of a targeted selective treatment (TST) as an alternative control approach of their parasitism. A longitudinal observational study was developed during a period of five years in a population of 62 Miranda donkeys. At first, strategic deworming of these animals was performed every semester, but this was progressively replaced by a TST approach according to the levels of Eggs per Gram (EPG). This new deworming strategy was conducted in association with a regular parasitological monitoring of the animals every three months, being dewormed with ivermectin when egg shedding was higher than 500 EPG. Over the study period, a decrease of the annual prevalence rate of infection by gastrointestinal strongyles was observed, from 35.5% to 19.4%, as well as a negative binomial distribution of parasitic strongyles in donkeys submitted to selective anthelminthic control. The prevalence rate of infection was higher in females (39.5%), in individuals under four years (46.7%) and in those presenting a lower body condition (40.8%). The egg output was higher in animals younger than four years (589.3 EPG) than in those older than 10 years (533.6 EPG) (p < 0.05). However, no differences were observed according to sex during the study period. Results from this study allowed to note the influence of swampy pastures and of the weather changes in the epidemiology of strongylosis in Miranda donkeys. Moreover, it was possible to establish the annual epidemiological curve of strongyle egg shedding, with June being the month with the highest EPG, December having the lowest EPG and March and September showing intermediate numbers. Overall, a lower biodiversity of gastrointestinal parasites was observed. Cyathostomum sensu lato was the most prevalent genus and Strongylus vulgaris was the most observed large strongyle of the Strongylidae family. Trichostrongylus axei and Parascaris sp. were other nematodes with a minor frequency. The higher prevalence of strongyles at the beginning of the study showed a progressive decrease throughout the research period, and also for parasite biodiversity. Therefore, a targeted selective treatment seems to be a rational anthelminthic control approach in Miranda donkey strongyle infection and in other gastrointestinal parasites, since it reduces the antiparasitic treatments, the parasite' prevalence and the EPG level. However, a loss of parasite biodiversity was noted at the end of the study period, as Cyathostominae were the only isolated strongyles. This can be a challenging situation in the long run, taking the ability of these nematodes to adapt easily to any deworming program, meaning that fecal EPG monitoring should be kept as a rule to a rational parasite control program.

First Molecular Characterization of Hypoderma actaeon in Cattle and Red Deer (Cervus elaphus) in Portugal.

Hypoderma spp. larvae cause subcutaneous myiasis in several animal species. The objective of the present investigation was to identify and characterize morphologically and molecularly the larvae of Hypoderma spp. collected from cattle (Bos taurus taurus) and red deer (Cervus elaphus) in the district of Castelo Branco, Portugal. For this purpose, a total of 8 larvae were collected from cattle (n=2) and red deer (n=6). After morphological identification of Hypoderma spp. larvae, molecular characterization was based on PCR-RFLP and mitochondrial CO1 gene sequence analysis. All larvae were morphologically characterized as the third instar larvae (L3) of H. actaeon. Two restriction enzymes were used for molecular identification of the larvae. TaqI restriction enzyme was not able to cut H. actaeon. However, MboII restriction enzyme differentiated Hypoderma species showing 210 and 450 bp bands in H. actaeon. Furthermore, according to the alignment of the mt-CO1 gene sequences of Hypoderma species and to PCR-RFLP findings, all the identified Hypoderma larvae were confirmed as H. actaeon. This is the first report of identification of Hypoderma spp. (Diptera; Oestridae) from cattle and red deer in Portugal, based on morphological and molecular analyses.

Prevalence of Coxiella burnetii antibodies in Portuguese dairy cattle herds.

Q fever is an important zoonotic disease which has been recently diagnosed, mainly in sheep and goats, in Portugal. The aim of the present study was to determine the prevalence of bovine Coxiella burnetii antibodies in dairy farms from the northwest of Portugal. Bulk tank milk samples were randomly obtained, on November 2013, from 90 dairy farms and assayed using an ELISA kit. The apparent prevalence was 61.1% (95% C.I. from 50.8 to 70.5%). The proportion of negative and intermediate (inconclusive) herds was 34.5% (25.5 to 44.7%) and 4.4% (1.7 to 10.9%), respectively. In conclusion, a high level of exposure to Coxiella burnetii was observed in Portuguese dairy cattle herds, highlighting the needs to better understand the epidemiology of Q fever in Portugal by the implementation of a monitoring program based on harmonized serologic and molecular methodologies and elucidation of the infection status of the herds.

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.