Dirofilaria repens in dogs and humans in Lithuania

Abstract

Background In Lithuania, the first case of canine subcutaneous dirofilariosis was recorded in 2010. Since then, an increasing number of cases of canine dirofilariosis have been documented in different veterinary clinics throughout the country. Human dirofilariosis was diagnosed in Lithuania for the first time in September 2011. However, to the authors’ knowledge, there are no published data on the presence and prevalence of autochthonous dirofilariosis in dogs and humans in the country. The present study provides information about the predominant species and prevalence of Dirofilaria in dogs and describes the cases of human dirofilariosis in Lithuania. It also outlines PCR detection of the bacterial endosymbiont Wolbachia that contributes to the inflammatory features of filarioid infection. Results A total of 2280 blood samples and six adult worms from pet and shelter dogs were collected in the central and eastern regions of Lithuania in 2013–2015. Based on their morphological appearance, morphometric measurements and molecular analysis, all the adult nematodes were identified as Dirofilaria repens. The diagnosis of microfilariae in blood samples was based on blood smear analysis and Knott’s test. The PCR and sequence analysis of the ribosomal DNA ITS2 region and cox 1 gene confirmed the presence of D. repens . Overall, 61 (2.7%) of the 2280 blood samples were found to be positive for the presence of D. repens. The infection rate of D. repens was significantly higher in shelter dogs (19.0%; 19/100) than in pet dogs (1.9%; 42/2180) ( χ 2 = 100.039, df = 1, P < 0.0001). Forty-nine DNA samples of D. repens -infected dogs were tested for the presence of the bacterial endosymbiont Wolbachia and, of these, 40 samples (81.6%) were found to be positive. Three ocular and six subcutaneous cases of human dirofilariosis were diagnosed in Lithuania in the period 2011–2018. Conclusions To the authors’ knowledge, this is the first report of autochthonous D. repens infection in dogs and humans in Lithuania. The present data demonstrate that D. repens is the main etiological agent of dirofilariosis in Lithuania. The DNA of the filarioid endosymbiotic bacterium Wolbachia was detected in the vast majority of dogs infected with D. repens .

Development of a multiplexed qPCRs-based approach for the diagnosis ofDirofilaria immitis, D. repens,Acanthocheilonema reconditumand the others filariosis

“…In addition, the occult heartworm cannot be confirmed by serological tests when presented as a co-infection with another filarial infection that cross-reacts with the test, especially if associated with D. repens infection (Fig. 2), this type of case resulting from an unknown mechanism of D. repens microfilariae, which shows a suppressive effect on those of D. immitis [38]. The ESDA does not recommend the routine heat-pretreatment of sera in area endemic for these parasitosis, as the only recommends it to resolve the discrepancy between the other tests, particularly when the dog is positive on the microfilaria test and negative by serology, and to confirm a suspicion of clinical disease suggestive of microfilaremia [11, 12].…”

“…These reactions were performed in a thermal cycler CFX96 Touch detection system (Bio-Rad, Marnes-la-Coquette, France) after activating the readers of the dyes used in each qPCR systems. After running protocol, the accumulation of the relative fluorescence units (RFUs) was recorded during the extension step of qPCRs and used to set-up the cut-off value of each TaqMan system, according to the formula described [38]. Here, we fixed the tolerance value at 5% for all systems.…”

“…The qPCR reaction was considered positive only if it showed a RFUs value higher than Cut Off. All these models were performed using the CFX Manager Software Version 3[38].…”

“…Furthermore, the specificity of multiplex qPCR systems was also assessed using individual and pooled DNA, where the DNA of D. immitis, D. repens and A. reconditum were used to validate the triplex, D. immitis and A. vasorum were used to validate duplex targeting heartworms and, finally, the wolbachiae DNA extracted from D. immitis and D. repens microfilariae were used as positive control to validate duplex system targeting these Wolbachia . The analytical sensitivity was assessed using a 10-fold dilution of DNA templates, and then standard curves and derived parameters (PCR efficiency, Slope, Y-intercept and correlation coefficient) were generated using CFX Manager Software Version 3 [38]. The triplex and pan-filarial qPCR systems were challenged in detecting the related microfilariae number of D. immitis , D. repens and A. reconditum .…”

Pathogen screening in the red fox (Vulpes Vulpes) from Lithuania

“…Dirofilariasis is recognized as a zoonosis spreading across Europe (Genchi et al, 2009;Genchi et al, 2011;Simón et al, 2012). A previous study showed that D. repens is a zoonotic parasite in Lithuania (Sabūnas et al, 2019a). In Lithuania, nine human cases of D. repens during the period of 2011-2018 and the prevalence of D. repens among shelter dogs have been reported.…”