Molecular epidemiology of Canine circovirus in domestic dogs and wildlife in Namibia, Africa.

Canine circovirus (CanineCV) is a DNA virus affecting domestic dogs and other wild carnivore species. Despite the potential implications for dogs' health and wildlife conservation, data on CanineCV presence, epidemiology and genetic features from Africa is still poor. In the present study, biological specimens collected between 2020 and 2022 from a total of 32 jackals and 575 domestic dogs were tested for the presence of CanineCV DNA to evaluate its frequency. Furthermore, sequencing was conducted on positive samples to characterize the strains and compare them with publicly available sequences through phylogenetic analysis. A high CanineCV prevalence was observed both in jackals (43.75%; 95 CI: 28.17% - 60.67%) and domestic dogs (27.13%; 95 CI: 23.66% - 30.91%). All aside from one Namibian strain formed an independent clade, suggestive of extremely rare introduction events, followed by local persistence, circulation, and evolution. Remarkably, different recombination events were observed involving strains from both jackals and domestic dogs, which testify to the likely strain exchange between these populations. Distinctive amino acid residues were also observed in jackals. The limitations of the considered host populations however prevent a definitive conclusion on host adaptation, biological, and clinical features. Further studies should be performed to expand our current knowledge of the CanineCV disease scenario in Namibia, other African regions, and associated host species in Africa.

Molecular epidemiology of canine parvovirus in Namibia: Introduction pathways and local persistence.

Canine parvovirus is a member of the Carnivore protoparvovirus 1 species that, after a relatively recent origin, has reached a worldwide distribution. Like other ssDNA viruses, it is featured by a remarkable evolutionary rate and thus genetic variability. CPV-2 is responsible for a severe systemic infection affecting especially domestic dogs. However, other carnivores, including wild species, are susceptible and thus represents a menace to wildlife conservation too. Despite the relevance of the topic, molecular epidemiology data are scarce and outdated in certain areas of the world, like Africa and, in particular, Namibia. The present study investigates the occurrence and genetic features of CPV in Namibian domestic dogs and jackals. The VP2 of detected strains was characterized and analyzed to assess the viral circulation and link among host species, Namibian districts and foreign countries. With the only exception of one New-CPV-2a, all the detected strains belonged to the CPV-2c antigenic variant and were closely related to strains of Asian origin. Nevertheless, a dedicated phylogeographic analysis revealed that the introduction was more likely mediated by other African countries, highlighting the challenge of controlling illegal animal imports across land borders. Similarly, the absence of any geographical clustering within Namibia testify a substantially unconstrained viral circulation among districts. The absence/incomplete vaccination status reported by the animal owners could have significantly contributed to the infection's success after its introduction. Finally, infection of a wild jackal was also proven. Although the limited wild animals' sample size prevents any definitive conclusion, the identity of the sequences from the jackal and the ones originating from the domestic dogs suggests a potential inter-species transmission. The epidemiological and clinical implications in wild specie remain obscure.

Prevalence and molecular characterization of Dirofilaria immitis in road killed canids of northern Iran.

BACKGROUND: Dirofilaria immitis is a mosquito-borne filarial nematode, which infects primarily wild and domestic canids, causing cardiopulmonary dirofilariasis. The aim of the present study was to determine the prevalence and characterize molecular features of D. immitis in road killed canids, northern Iran. METHODS: The carcasses of 53 road killed canids including 18 dogs (Canis familiaris), and 35 golden jackals (C. aureus) were necropsied in both Mazanderan and Guilan provinces, northern Iran. The molecular analyses were conducted based on the cytochrome oxidase (Cox) 1 and 18S ribosomal RNA (rRNA) genes. RESULTS: The heartworm infection was found in 55.6% of dogs and 22.9% of jackals. Our study revealed significantly higher prevalence of D. immitis in dogs compared to jackals (P = 0.031). The prevalence of D. immitis was no statistically significant between males and females in both dogs and jackal (P > 0.05). Comparison of the Cox1 gene sequences with available data in the GenBank illustrated 100% similarity with D. immitis isolates from different hosts in European, Asian, and South American continents. Moreover, the 18S rRNA gene sequences showed 100% identity with dog isolates from Japan and French Guiana. CONCLUSIONS: This study confirms the high prevalence of D. immitis in dogs and jackals of northern Iran. Developing control programs to prevent transmission of the disease is necessary for dogs and humans in the study areas.

Molecular Detection and Genetic Characterization of Porcine Circovirus 2 (PCV-2) in Black-Backed Jackal (Lupulella mesomelas) in Namibia.

Members of the genus Circovirus have been identified in several host species. While initially considered host-specific, there is growing evidence of their presence in unexpected hosts. Porcine circovirus 2 (PCV-2) is no exception, having been reported in animals belonging to different orders, including carnivores. Recently, PCV-2 was detected in domestic pigs, warthogs and antelopes in Namibia. Considering the potential contact between these populations and wild carnivores, the presence of PCV-2 was investigated in 32 black-backed jackals (Lupulella mesomelas) shot between February and July 2021 as part of predator control operations in Namibia. Two lung lymph nodes tested positive for PCV-2 by PCR, confirming the broader-than-expected PCV-2 host tropism and the susceptibility of canids. Sequence analysis demonstrated a close association between the PCV-2s identified in the jackals and PCV-2b strains collected from South African domestic pigs. Although several hypotheses regarding the source of the jackal's infection are proposed, further studies should be performed to properly assess how PCV-2 is acquired and maintained in the wild and its potential impact on wild and domestic species.

Positive selection on the MHC class II DLA-DQA1 gene in golden jackals (Canis aureus) from their recent expansion range in Europe and its effect on their body mass index.

BACKGROUND: In Europe, golden jackals (Canis aureus) have been expanding their range out of the southern and southeastern Balkans towards central Europe continually since the 1960s. Here, we investigated the level of functional diversity at the MHC class II DLA-DQA1 exon 2 in golden jackal populations from Bulgaria, Serbia, and Hungary. Specifically, we tested for positive selection on and geographic variation at that locus due to adaptation to supposedly regionally varying pathogenic landscapes. To test for potential fitness effects of different protein variants on individual body condition, we used linear modeling of individual body mass indexes (bmi) and accounted for possible age, sex, geographical, and climatic effects. The latter approach was performed, however, only on Serbian individuals with appropriate data. RESULTS: Only three different DLA-DQA1 alleles were detected, all coding for different amino-acid sequences. The neutrality tests revealed no significant but positive values; there was no signal of spatial structuring and no deviation from the Hardy-Weinberg equilibrium across the studied range of expansion. However, we found a signal of trans-species polymorphism and significant test results for positive selection on three codons. Our information-theory based linear modeling results indicated an effect of ambient temperature on the occurrence of individual DLA-DQA1 genotypes in individuals from across the studied expansion range, independent from geographical position. Our linear modeling results of individual bmi values indicated that yearlings homozygous for DLA-DQA1*03001 reached values typical for adults contrary to yearlings carrying other genotypes (protein combinations). This suggested better growth rates and thus a possible fitness advantage of yearlings homozygous for DLA-DQA1*03001. CONCLUSIONS: Our results indicate a demographic (stochastic) signal of reduced DLA-DQA1 exon 2 variation, in line with the documented historical demographic bottleneck. At the same time, however, allelic variation was also affected by positive selection and adaptation to varying ambient temperature, supposedly reflecting geographic variation in the pathogenic landscape. Moreover, an allele effect on body mass index values of yearlings suggested differential fitness associated with growth rates. Overall, a combination of a stochastic effect and positive selection has shaped and is still shaping the variation at the studied MHC locus.

Black-backed jackals (Canis mesomelas) from semi-arid rangelands in South Africa harbour Hepatozoon canis and a Theileria species but apparently not Babesia rossi.

Despite the importance of disease as a wildlife management challenge in South Africa, baseline data on the epidemiology of pathogens occurring in free-ranging species has received little attention to date. Black-backed jackals (Canis mesomelas) are a wide-ranging, abundant carnivore with substantial economic importance due to their role in livestock depredation. They are known reservoirs hosts of Babesia rossi, a virulent pathogen in domestic dogs in sub-Saharan Africa. We investigated the prevalence and diversity of tick-borne pathogens (TPBs) including Babesia, Theileria, Hepatozoon, Ehrlichia and Anaplasma species, together with host-attached tick diversity, in a black-backed jackal population from the semi-arid Central Karoo, a small-livestock farming region in South Africa. Using reverse line blot hybridisation, we screened 43 blood samples and sequenced the 18S rRNA gene from positive samples to confirm and characterise pathogen identity using a phylogenetic framework. Hepatozoon canis, a ubiquitous pathogen of domestic and wild canids globally, was observed in 47% of jackals, while a Theileria sp. most similar to T. ovis, a piroplasm found in small livestock, was observed in 5% of jackals. No Babesia, Ehrlichia or Anaplasma species were identified, although a Sarcocystis sp. sequence was isolated from one jackal. Host-attached ticks (n = 20) comprised three species, Amblyomma marmoreum, Haemaphysalis elliptica/zumpti and Ixodes rubicundus, commonly known ticks in the region. In summary, prevalence of TBPs in black-backed jackals from this semi-arid rangeland region was lower than in jackal populations in more mesic regions. These jackals were apparently not infected with B. rossi. While this study is one of the first investigations into the epidemiology of TBPs infecting jackals and adds to the sparse literature, further studies which span landscape uses, climate conditions and seasonality are encouraged.

Subcutaneous ticks: a first report in a golden jackal, and their absence in non-canid carnivores.

BACKGROUND: Ticks are hematophagous arthropods which normally attach to the surface of the host's skin. Their aberrant presence in the subcutaneous tissue of a few carnivores, predominantly foxes, has been reported. However, there have been no reports of this phenomenon in other carnivores such as mustelids or golden jackals. Our aim was to investigate the host spectrum for this aberrant localization of ticks. METHODS: Between 2015 and 2020, a total of 198 carcasses of 12 species of carnivore were examined by parasitological necropsy. When a subcutaneous tick was found, the nodule was removed, carefully dissected, and stored in ethanol. The morphological identification of the subcutaneous tick was carried out to species level. RESULTS: A single subcutaneous tick was found in one carcass, that of a golden jackal (Canis aureus). The tick was identified as a female Ixodes ricinus. All the other carcasses were negative for the presence of subcutaneous ticks. CONCLUSION: To our knowledge, this is the first report of a subcutaneous tick in a golden jackal. This finding broadens the host spectrum of subcutaneous ticks, and reinforces the idea that, among carnivores, this phenomenon only occurs in canids.

Rapid and Reliable Detection of Echinococcus multilocularis from Faeces Using Droplet Digital PCR.

PURPOSE: Alveolar echinococcosis is a severe helminthic disease in humans caused by larvae of the fox tapeworm Echinococcus multilocularis. Austria is considered an endemic area with hotspots having up to 45% prevalence (Bagó et al. in Proceedings of the Zoo and Wildlife Health Conference 2019, Berlin, p. 91, 2019). At our facility, we have registered a notifiable increase of animals submitted for the diagnosis of E. multilocularis since 2016. Therefore, we investigated high throughput diagnostic methods to provide rapid and reliable results in comparison with our current method. METHODS: We have developed and compared a novel method of detection using droplet digital PCR (ddPCR) combined with previous target specific extraction according to Maas et al. (Vet Parasitol 230:20-24, 2016), with our current macroscopic method "Shaking in a Vessel Technique" (SVT) by Duscher et al. (Parasitol Res 95(1):40-42, 2005). We investigated 77 wild canids (72 red foxes, 5 golden jackals) using both methods. The data were analyzed using a non-Bayesian approach, applying bootstrapping to create confidentiality intervals. RESULTS: Sensitivity for droplet digital PCR was 90.51% with the 95% credibility interval ranging from 82.50 to 96.92%, whereas mean sensitivity for SVT was 92.04% with a 95% credibility interval ranging from 84.75% to 98.36%. Additionally, a non-linear regression similar to R2 could be pointed out between the counted worms and the results gathered from ddPCR. CONCLUSION: Magnetic capture extraction followed by ddPCR shows strong potential as a high throughput method for diagnosing E. multilocularis prevalence in diverse canid populations as well as infection intensities of individual animals, giving valuable epidemiological insights of the distribution amongst wild canids as an alternative to conventional qPCR or macroscopic methods.

Molecular survey of Dirofilaria species in stray dogs, red foxes and golden jackals from Vojvodina, Serbia.

Cardiopulmonary dirofilariosis in dogs and other carnivores is caused by Dirofilaria immitis, while Dirofilaria repens usually causes a subcutaneous infection. The importance of red foxes and golden jackals in the epidemiology of dirofilariosis remains unknown. Thus, the aim of this study was to conduct a cross-sectional molecular survey of Dirofilaria species in stray dogs, red foxes and golden jackals from the endemic region of Vojvodina, Serbia, in order to determine and update data on their prevalence and provide insight into the epidemiological importance of wild canids. A total of 59 blood samples from stray dogs, 94 from red foxes and 32 from golden jackals were collected and screened by real-time PCR targeting a 115-bp fragment of the mitochondrial 12S gene of filarioids and by conventional PCR assay targeting a 484-524-bp fragment of 5.8S-ITS2-28S locus of filarioids. The cross-sectional molecular survey detected the filarioid mitochondrial 12S gene fragment in stray dogs (27.1 %), red foxes (8.5 %) and golden jackals (6.3 %) in the same endemic region of Vojvodina, Serbia. Only D. immitis was detected in stray dogs, while both D. immitis and D. repens were detected in populations of red foxes and golden jackals. These results outline a possible interaction of D. immitis infection between the dog population and the wild canid populations, while D. repens was found to circulate mostly in golden jackals and red foxes populations.

High prevalence of vector-borne pathogens in domestic and wild carnivores in Iraq.

Vector-borne diseases (VBDs) of domestic and wild carnivores are of major public health concern both in industrialized and developing countries, especially in poor socioeconomic settings. War-torn areas specifically suffer from absence of veterinary surveillance of VBDs, resulting in lack of scientific knowledge on this topic. To investigate occurence and prevalence of several vector-borne pathogens (VBPs) in some carnivore species from Iraq, blood samples (n = 397) were obtained from 190 canids [97 stray dogs (Canis familiaris), 55 jackals (Canis aureus) and 38 red foxes (Vulpes vulpes)] and 207 stray cats (Felis catus) collected during a feral animal control and zoonotic disease surveillance program in several United States military bases in Iraq. The presence of Babesia spp., Hepatozoon spp., Ehrlichia spp., Anaplasma spp., Dirofilaria spp. and Leishmania spp. DNA was molecularly investigated. Out of 397 animals tested, 176 (44.3%; 95% CI: 39.5-49.2%) were positive for at least one pathogen with the highest prevalence in foxes (73.7%; 95% CI: 58-85%), followed by jackals (54.5%; 95% CI: 41.5-67%), dogs (38.1%; 29.1-48.1%) and cats (39.1%; 95% CI: 32.7-45.9%). Up to five pathogens were diagnosed in dogs. Hepatozoon canis was the most prevalent VBP in jackals (49.1%; 95% CI: 36.4-61.9%), foxes (47.3%; 95% CI: 32.5-62.7%) and dogs (33%; 95% CI: 24.4-42.8%), whereas Hepatozoon felis was the only species detected in cats (39.1%; 95% CI: 32.7-45.9%). A species of Babesia related to but different from Babesia lengau and designated as Babesia sp. MML was detected in six foxes (15.8%; 95% CI: 7.4-30.4%) and in one jackal (1.8%; 95% CI: 0.3-9.6%). This finding suggested the existence of a new species in the genus Babesia as inferred by molecular and phylogenetical analysis. Further, Babesia vulpes was identified only in two foxes (5.3%; 95% CI: 1.5-17.3%). All samples were negative for Leishmania spp. and Ehrlichia spp. Co-infection with H. canis and Babesia spp. was the most prevalent (5/176, 2.8%, i.e., 4 foxes and 1 jackal), followed by H. canis and Dirofilaria immitis (1/176, 1.3%, i.e., in 1 jackal), H. canis and Dirofilaria repens or Acanthocheilonema reconditum (1/176, 1.3%, i.e., in one dog, each). Data presented fill gaps into knowledge of VBPs in dogs, cats and wild canids in Iraq, indicating that different pathogens circulate amongst animal populations living in the same areas, possibly sharing the same tick vectors. Large-scale surveys are urgently needed to further assess VBPs distribution in Iraq and establish preventative strategies in domestic animals to minimize the risk of infection for animals and humans.