Epizootiology and Pathology of Sarcoptic Mange in Two Species of Fox (Lycalopex spp.) in Human-Dominated Landscapes of Central Chile.

Sarcoptic mange caused by Sarcoptes scabiei can have catastrophic consequences for wildlife. We inspected 122 Andean foxes (Lycalopex culpaeus), collected by active (n=66) or passive (n=56) surveillance, and 28 South American gray foxes (Lycalopex griseus; all from passive surveillance) for mange in Chile (2015-19). In Andean foxes, gross lesions of mange were diagnosed in 24% of passively and 9% of actively collected foxes, although observed prevalences might be underestimated. Seroprevalence was 37 and 18%, respectively, indicating that some individuals recovered from infection or were developing the disease. No differences were found between age and sex groups. Comparing data from passive surveillance, occurrence of gross lesions was lower in gray foxes (5%). Body condition was significantly better in Andean foxes without lesions than in diseased foxes, which had significantly lower albumin concentrations than healthy individuals. Among the 12 foxes with gross lesions, four, six and two individuals were categorized as having type I, type II, and type III lesions, respectively, based on clinical severity. Histologic severity correlated with gross lesions and included irregular epidermal hyperplasia with hyperkeratosis, which was marked in type II and III infections. Conventional PCR targeting of the cox1 gene fragment revealed four nucleotide sequence types, showing 99-100% identity among them and between 99% and 100% identity with previously published sequences of S. scabiei. A significant association between the occurrence of mange in foxes and distance to the nearest house was found. We speculate that diseased foxes tended to approach human settlements, perhaps in search of food. Visual inspection of 211 rural dogs from the study area did not reveal gross mange lesions in any animal. Sarcoptic mange is enzootic in the Andean fox in the study area and should be considered in the management of the species.

Molecular identification of Bartonella spp. and Rickettsia felis in fox fleas, Chile.

Seventy-five flea pools (one to ten fleas per pool) from 51 Andean foxes (Lycalopex culpaeus) and five South American grey foxes or chillas (Lycalopex griseus) from the Mediterranean region of Chile were analyzed for the presence of DNA of Bartonella spp. and Rickettsia spp. through quantitative real-time PCR for the nouG and gltA genes, respectively. Positive samples were further characterized by conventional PCR protocols, targeting gltA and ITS genes for Bartonella, and gltA, ompA, and ompB genes for Rickettsia. Bartonella was detected in 48 % of the Pulex irritans pools (B. rochalimae in three pools, B. berkhoffii in two pools, B. henselae in one pool), and 8 % of the Ctenocephalides felis felis pools (B. rochalimae, one pool). Rickettsia was confirmed in 11 % of P. irritans pools and 92 % of the Ct. felis pools. Characterization confirmed R. felis in all sequenced Rickettsia-positive pools. All Ct. canis pools were negative. A Ct. felis pool from a wild-found domestic ferret (Mustela putorius furo) also resulted positive for R. felis. Although opportunistic, this survey provides the first description of zoonotic pathogens naturally circulating in fleas parasitizing Chilean free-living carnivores.

Spirocerca lupi in the stomach of two Andean foxes (Lycalopex culpaeus) from Chile.

The genus Spirocerca includes nematodes that parasitize the stomach and the oesophagus of carnivores, chiefly canids. Herein, we provide new data about the morphological, histopathological, and molecular characterization of Spirocerca sp. in Andean foxes (Lycalopex culpaeus) in Chile. Intact immature worms, identified as Spirocerca sp., were recovered in the lumen of the stomach from two foxes. Histologically, worms morphologically consistent with spirurid nematodes were present within the wall of the stomach and surrounded by nodular areas of inflammation with central necrotic debris. Molecular analysis of the cox1 gene yielded 19 sequences and 5 nucleotide sequence types with 99.95 to 99.98% similarity, being shared between both foxes. Nucleotide similarity ranged from 93.1 (with genotype 2 of S. lupi and S. vulpis) to 95.8% (with genotype 1 of S. lupi), a higher similarity than noted from sequences of S. lupi from an Andean fox from Peru (91.0 to 93.3%). However, the Poisson Tree Processes for species delineation did not support the existence of a new species Spirocerca. Phylogenetic and nucleotide analyses suggest that these specimens belong to a new variant or genotype of S. lupi or to a cryptic species. Whether the presence of the worms in the stomach has to do with genotypic differences in parasites or host or some combination is uncertain. Spirocerca lupi has never been found in Chilean dogs and must be investigated.

Co-infection patterns of vector-borne zoonotic pathogens in owned free-ranging dogs in central Chile.

We investigated the co-occurrence of the nine of the most relevant canine vector-borne pathogens (CVBP) using conventional and real-time PCR and evaluated risk factors and potential non-apparent haematological alterations associated with co-infection in 111 rural, owned, free-ranging dogs in the Metropolitan Region of Chile.At least one pathogen was detected in 75% of the dogs. DNA of Anaplasma platys (Ap; 36%), Candidatus Mycoplasma haematoparvum (CMhp; 31%), Mycoplasma haemocanis (Mhc; 28%), Trypanosoma cruzi (17%), Leishmania spp. (4.5%), and Acanthocheilonema reconditum (1%) was detected. All dogs were negative for Ehrlichia spp., Rickettsia spp., Bartonella spp., Piroplasmida, and Hepatozoon spp. Thirty-eight dogs (34%) were coinfected. CMhp was involved in 71%, Mhc in 58%, and Ap in 50% of the co-infections. The most common co-infection pattern was CMhp-Mhc (37% of the cases). The prevalence of Ap was higher in juvenile than in adult dogs, whereas the opposite was found for CMhp and Mhc. Adult dogs were four times more likely of being co-infected than juveniles. Co-infected animals showed higher white blood cell count, segmented neutrophil count, and GGT levels than non-co-infected dogs. Clinically healthy but infected dogs may act as reservoirs of CVBP, and their free-ranging behavior would facilitate the spread of these pathogens to other dogs as well as human beings or wild carnivores.

SEROSURVEY FOR SELECTED PARASITIC AND BACTERIAL PATHOGENS IN DARWIN'S FOX (LYCALOPEX FULVIPES): NOT ONLY DOG DISEASES ARE A THREAT.

The Darwin's fox (Lycalopex fulvipes) is one of the most endangered carnivores worldwide, with the risk of disease spillover from domestic dogs being a major conservation threat. However, lack of epidemiologic information about generalist, non-dog-transmission-dependent protozoal and bacterial pathogens may be a barrier for disease prevention and management. To determine the exposure of some of these agents in Darwin's fox populations, 54 serum samples were collected from 47 Darwin's foxes in Southern Chile during 2013-18 and assessed for the presence of antibodies against Brucella abortus, Brucella canis, Coxiella burnetii, pathogenic Leptospira (serovars Grippotyphosa, Pomona, Canicola, Hardjo, and Copehageni), Toxoplasma gondii, and Neospora caninum. The highest seroprevalence was detected for T. gondii (78%), followed by pathogenic Leptospira (14%). All the studied Leptospira serovars were confirmed in at least one animal. Two foxes seroconverted to Leptospira and one to T. gondii during the study period. No seroconversions were observed for the other pathogens. No risk factors, either intrinsic (sex, age) or extrinsic (season, year, and degree of landscape anthropization), were associated with the probability of being exposed to T. gondii. Our results indicate that T. gondii exposure is widespread in the Darwin's fox population, including in areas with minimal anthropization, and that T. gondii and pathogenic Leptospira might be neglected threats to the species. Further studies identifying the causes of morbidity and mortality in Darwin's fox are needed to determine if these or other pathogens are having individual or population-wide effects in this species.

Widespread Infection with Hemotropic Mycoplasmas in Free-Ranging Dogs and Wild Foxes Across Six Bioclimatic Regions of Chile.

Blood samples of 626 rural dogs, 140 Andean foxes (Lycalopex culpaeus), and 83 South American grey foxes (L. griseus) from six bioregions of Chile spanning 3000 km were screened for Mycoplasma DNA by conventional PCR and sequencing. Risk factors of infection were inferred using Generalized Linear Mixed Models and genetic structure by network analyses. Overall, Mycoplasma haemocanis/Mycoplasma haemofelis (Mhc/Mhf) and Candidatus Mycoplasma haematoparvum (CMhp) observed prevalence was 23.8% and 12.8% in dogs, 20.1% and 7.2% in Andean foxes, and 26.5% and 8.4% in grey foxes, respectively. Both hemoplasmas were confirmed in all the bioregions, with higher prevalence in those where ticks from the Rhipicephalus sanguineus species group were absent. Candidatus M. haematominutum and a Mycoplasma sp. previously found in South American carnivores were detected in one fox each. Although the most prevalent Mhc/Mhf and CMhp sequence types were shared between dogs and foxes, network analysis revealed genetic structure of Mhc/Mhf between hosts in some regions. Male sex was associated with a higher risk of Mhc/Mhf and CMhp infection in dogs, and adult age with CMhp infection, suggesting that direct transmission is relevant. No risk factor was identified in foxes. Our study provides novel information about canine hemoplasmas with relevance in distribution, transmission routes, and cross-species transmission.

Serological Survey and Molecular Characterization of Theileria annulata in Sicilian Cattle.

Tropical theileriosis is a tick-borne disease caused by hemoprotozoan parasites with considerable veterinary and economic impact worldwide. Ticks transmitting the disease belong to the Haemaphysalis, Rhipicephalus, and Hyalomma genera. The Hyalomma genus is very common in Sicily (Italy) and represents the main Theileria annulata vector in the island. Data concerning the molecular epidemiology of this pathogen are missing in the region. In 2018-2019, blood and serum samples were collected from 480 cows in seven Sicilian farms from four different provinces. Seroprevalence in the farms ranged from 22% to 71%. Three farms were selected for molecular analysis consisting of real-time PCR targeting the almost complete 18S ribosomal RNA (rRNA). Four amplicons per farm were sequenced and phylogenetic analyses were carried out. The four sequences were identical within each farm and showed 92-99% identity with the other farms and with sequences from Genbank. According to the phylogenetic analysis, these three sequences and an additional one from a laboratory-cultured Theileria annulata strain obtained in 1999 belonged to a single T. annulata clade with good bootstrap support with other sequences from Italy, India, and Iran, indicating limited geographical and temporal genetic variability of the parasite. This study represents the first phylogenetic analysis of T. annulata in Sicily, which will be useful to improve the strategies for theileriosis control and prevention.

Worldwide occurrence of haemoplasmas in wildlife: Insights into the patterns of infection, transmission, pathology and zoonotic potential.

Haemotropic mycoplasmas (haemoplasmas) have increasingly attracted the attention of wildlife disease researchers due to a combination of wide host range, high prevalence and genetic diversity. A systematic review identified 75 articles that investigated haemoplasma infection in wildlife by molecular methods (chiefly targeting partial 16S rRNA gene sequences), which included 131 host genera across six orders. Studies were less common in the Eastern Hemisphere (especially Africa and Asia) and more frequent in the Artiodactyla and Carnivora. Meta-analysis showed that infection prevalence did not vary by geographic region nor host order, but wild hosts showed significantly higher prevalence than captive hosts. Using a taxonomically flexible machine learning algorithm, we also found vampire bats and cervids to have greater prevalence, whereas mink, a subclade of vesper bats, and true foxes all had lower prevalence compared to the remaining sampled mammal phylogeny. Haemoplasma genotype and nucleotide diversity varied little among wild mammals but were marginally lower in primates and bats. Coinfection with more than one haemoplasma species or genotype was always confirmed when assessed. Risk factors of infection identified were sociality, age, males and high trophic levels, and both prevalence and diversity were often higher in undisturbed environments. Haemoplasmas likely use different and concurrent transmission routes and typically display enzootic dynamics when wild populations are studied longitudinally. Haemoplasma pathology is poorly known in wildlife but appears subclinical. Candidatus Mycoplasma haematohominis, which causes disease in humans, probably has it natural host in bats. Haemoplasmas can serve as a model system in ecological and evolutionary studies, and future research on these pathogens in wildlife must focus on increasing the geographic range and taxa of studies and elucidating pathology, transmission and zoonotic potential. To facilitate such work, we recommend using universal PCR primers or NGS protocols to detect novel haemoplasmas and other genetic markers to differentiate among species and infer cross-species transmission.

Molecular investigation of zoonotic intracellular bacteria in Chilean bats.

Intracellular pathogens were investigated for the first time in 55 Chilean bats belonging to six species. Using a conventional PCR protocol targeting a fragment of the ITS region, 21 bats (38 %) were positive for DNA of Bartonella sp. Molecular characterization of fragments of the gltA, rpoB and fstZ genes and subsequent phylogenetic analysis indicated the presence of diverse genotypes related to Bartonella from bats worldwide. DNA from C. burnetii was investigated using a real-time PCR (qPCR) protocol targeting the IS1111 gene and yielded positive results for 5 individuals (9%), being the first report of C. burnetii in wildlife in Chile. All bats were negative for Rickettsia sp., evaluated by qPCR for the gltA gene, confirming that bats do not act as important reservoirs for Rickettsia. This preliminary survey calls for more comprehensive studies on the epidemiology of these agents, including larger sample sizes, the evaluation of potential transmission routes and spillover potential.

Babesia vogeli in dogs in Chile.

We report the presence of Babesia vogeli in dogs in Chile. During two surveillance campaigns separated by a year, sixty-three blood samples from free-ranging rural dogs in Coquimbo, Chile, were analysed through conventional PCR screening of the 18S rRNA for Babesia species. Sequencing confirmed the presence of Babesia vogeli in 6.3% of the tested animals, with positive cases in both years. All the sequences showed 100% nucleotide sequence identity among them and 99.8% with other previously published sequences from dogs. No clinical signs or haematological abnormalities other than thrombocytopenia were found in the parasitized individuals. This is the first report of a canine piroplasmid in Chile.

Hemoplasmas Are Endemic and Cause Asymptomatic Infection in the Endangered Darwin's Fox (Lycalopex fulvipes).

Mycoplasma haemocanis is prevalent in the endangered Darwin's fox (Lycalopex fulvipes) in its main stronghold, Chiloé Island (Chile). The origin of the infection, its dynamics, its presence in other fox populations and the potential consequences for fox health remain unexplored. For 8 years, hemoplasmal DNA was screened and characterized in blood from 82 foxes in Chiloé and two other fox populations and in 250 free-ranging dogs from Chiloé. The prevalence of M. haemocanis in foxes was constant during the study years, and coinfection with "Candidatus Mycoplasma haematoparvum" was confirmed in 30% of the foxes. Both hemoplasma species were detected in the two mainland fox populations and in Chiloé dogs. M. haemocanis was significantly more prevalent and more genetically diverse in foxes than in dogs. Two of the seven M. haemocanis haplotypes identified were shared between these species. Network analyses did not show genetic structure by species (foxes versus dogs), geographic (island versus mainland populations), or temporal (years of study) factors. The probability of infection with M. haemocanis increased with fox age but was not associated with sex, season, or degree of anthropization of individual fox habitats. Some foxes recaptured years apart were infected with the same haplotype in both events, and no hematological alterations were associated with hemoplasma infection, suggesting tolerance to the infection. Altogether, our results indicate that M. haemocanis is enzootic in the Darwin's fox and that intraspecific transmission is predominant. Nevertheless, such a prevalent pathogen in a threatened species represents a concern that must be considered in conservation actions.IMPORTANCEMycoplasma haemocanis is enzootic in Darwin's foxes. There is a higher M. haemocanis genetic diversity and prevalence in foxes than in sympatric dogs, although haplotypes are shared between the two carnivore species. There is an apparent tolerance of Darwin's foxes to Mycoplasma haemocanis.

Antimicrobial resistance genes in Andean foxes inhabiting anthropized landscapes in central Chile.

Antimicrobial resistance (AMR) is considered an emerging public health problem. Greater AMR development rate is associated with "antibiotic-using" environments. Wildlife thriving in anthropized landscapes could be good indicators of the burden of AMR and antibiotic resistance genes (ARGs) in these areas. The aim of this study was to determine the presence and load of ARGs in fecal swabs of wild Andean foxes (Lycalopex culpaeus) from anthropized landscapes of central Chile. DNA was extracted from samples of 72 foxes; 22 ARGs encoding resistance against 8 antibiotic groups were evaluated using qPCR. Eighteen of the 22 ARGs were found and tet(Q) (65.3%; 15/72 of the samples) was the most common gene detected. Almost half of the foxes presented a 'multiresistant microbiome' (i.e. at least three ARG encoding resistance to different groups of antimicrobials). Prevalence of tet(Q) was higher in the cold-humid season than in the warm-dry season, but not for other genes. Up to 15 and 13 ARGs were detected in the fecal samples from two additional foxes that were kept 6 and 11 days, respectively, in a clinical environment (Wildlife Rescue Center) and received antibiotic treatment. Some of the ARGs detected (e.g. mecA and blaCTX-M) in the present study are of particular concern from the public health perspective. Wild foxes seem to be good sentinels for ARG environmental burden in highly anthropized environments of central Chile.

Hemotropic mycoplasmas in bats captured near human settlements in Nigeria.

The presence of DNA of hemotropic mycoplasmas (hemoplasmas) was investigated for the first time in bats in Africa. Blood samples from 90 bats captured within or near human settlements in nine study areas from five states in Nigeria belonging to six genera of the families Pteropodidae, Rhinolophidae, and Molossidae were analyzed using conventional PCR protocol targeting a 391 bp part of the 16S rRNA gene. Of these, 32 samples (35 %) resulted positive. Eight nucleotide sequence types were identified, which were assigned to five genotypes showing between 93-99 % similarity with hemoplasmas from bats and/or rodents from other parts of the world, and/or Candidatus Mycoplasma haemohominis from a human patient. Network analysis showed genetic structure of hemoplasma sequences among bat species, but some sequences were shared among bats of different taxonomic groups and distant study areas. Further characterization of the samples using a protocol targeting ∼1200 bp of the 16S rRNA gene resulted in seven sequences that confirmed the results of the screening protocol. Hemoplasmas in Nigerian bats are prevalent, widely distributed and genetically diverse. The zoonotic risk to local inhabitants should not be neglected, due to the high similarity of some of the retrieved sequences with the human pathogen C. M. haemohominis.

An Eight-Year Survey for Canine Distemper Virus Indicates Lack of Exposure in the Endangered Darwin's Fox (Lycalopex fulvipes).

No evidence of exposure to canine distemper virus (CDV) was detected in 70 samples corresponding to 58 wild-trapped Darwin's foxes (Lycalopex fulvipes) in Chile. Given its current endangered status and it being immunologically naïve, in the event of a CDV spillover from dogs to foxes, high population mortality is expected.

Molecular Detection of Vector-Borne Pathogens in Rural Dogs and Associated Ctenocephalides felis Fleas (Siphonaptera: Pulicidae) in Easter Island (Chile).

The presence of vector-borne pathogens of veterinary and public health interest have received little attention in Chile. In Easter Island, in particular, a Chilean territory in the southeastern Pacific Ocean, no information is available. To fill this gap, 153 rural dogs were inspected for ectoparasites during a sterilization campaign carried out in 2016. Fleas were observed in 46% of the dogs, and Ctenocephalides felis (Bouché, 1835) was the only species present. Morphological identification of fleas was genetically confirmed using conventional polymerase chain reaction targeting the cox2 gene. No tick was observed in any dog. The presence of DNA of Rickettsia sp. (gltA and ompA fragment genes), Anaplasmataceae (16S rRNA), and Bartonella sp. (16S-23S ribosomal RNA intergenic spacer) was investigated in blood samples of 70 of the dogs and in 126 fleas analyzed in 68 pools that included 1-5 fleas. Rickettsial DNA was detected in 97% (n = 66) of the flea pools. Of these, 57 showed between 99 and 100% identity for both genes with published sequences of Candidatus Rickettsia asemboensis (CRa), six with Rickettsia felis, and one with Candidatus Rickettsia senegalensis. For two pools, gltA amplicons were identical to CRa but ompB amplicions showed 99-100% identity with R. felis. Anaplasmataceae DNA was detected in 16% (n = 11) pools. Sequenced amplicons showed highest identity with the endosymbiont Wolbachia pipientis. Bartonella DNA, showing 99% identity to Bartonella clarridgeiae, was detected in one pool (1.4%). No positive reaction was observed for any dog. This is the first detection of members of the 'R. felis-like' group other than R. felis in Chile.

Analysis of climate and extrinsic incubation of Dirofilaria immitis in southern South America.

Dirofilariosis, caused by Dirofilaria immitis and D. repens, is spreading in several geographic regions. The development of infective larvae in the mosquito vector (extrinsic incubation) needs an accumulated total of 130 degree-days above the 14 °C threshold, normally expressed as heartworm development units (HDUs). Based on this information, temperature- based models have been developed and applied to evaluate the distribution and spread of Dirofilaria infections in various countries and continents. Despite the confirmed presence of D. immitis in most South American countries, the available information about its epidemiology remains scarce. We analysed the temporal and spatial extrinsic incubation of this parasite in Argentina, Chile and Uruguay, taking into account daily temperatures from 49 meteorological stations during a 30-year period (1982-2012). The theoretically possible number of D. immitis generations was calculated based on the number of meteorological stations that reached the 130-HDUs threshold. The resulting information was spatially interpolated using the inverse weighted distance (IWD) model to produce thematic maps. The model shows that 41 of the meteorological stations reach the threshold needed and that D. immitis transmission is markedly seasonal with a peak in late spring (December), stable during summer (January to March) and declining in the autumn (April and May). Suitable temperatures exist in Uruguay and most of Argentina, whereas D. immitis transmission in Chile is only possible in the north and in the central inlands. The results suggest that the climatic impact on D. immitis transmission must have been minimal in the countries investigated since the annual meteorological records did not change much during the 30-year period analysed.