Genetic Diversity and Connectivity of Southern Right Whales (Eubalaena australis) Found in the Brazil and Chile-Peru Wintering Grounds and the South Georgia (Islas Georgias del Sur) Feeding Ground.

As species recover from exploitation, continued assessments of connectivity and population structure are warranted to provide information for conservation and management. This is particularly true in species with high dispersal capacity, such as migratory whales, where patterns of connectivity could change rapidly. Here we build on a previous long-term, large-scale collaboration on southern right whales (Eubalaena australis) to combine new (nnew) and published (npub) mitochondrial (mtDNA) and microsatellite genetic data from all major wintering grounds and, uniquely, the South Georgia (Islas Georgias del Sur: SG) feeding grounds. Specifically, we include data from Argentina (npub mtDNA/microsatellite = 208/46), Brazil (nnew mtDNA/microsatellite = 50/50), South Africa (nnew mtDNA/microsatellite = 66/77, npub mtDNA/microsatellite = 350/47), Chile-Peru (nnew mtDNA/microsatellite = 1/1), the Indo-Pacific (npub mtDNA/microsatellite = 769/126), and SG (npub mtDNA/microsatellite = 8/0, nnew mtDNA/microsatellite = 3/11) to investigate the position of previously unstudied habitats in the migratory network: Brazil, SG, and Chile-Peru. These new genetic data show connectivity between Brazil and Argentina, exemplified by weak genetic differentiation and the movement of 1 genetically identified individual between the South American grounds. The single sample from Chile-Peru had an mtDNA haplotype previously only observed in the Indo-Pacific and had a nuclear genotype that appeared admixed between the Indo-Pacific and South Atlantic, based on genetic clustering and assignment algorithms. The SG samples were clearly South Atlantic and were more similar to the South American than the South African wintering grounds. This study highlights how international collaborations are critical to provide context for emerging or recovering regions, like the SG feeding ground, as well as those that remain critically endangered, such as Chile-Peru.

Molecular survey for cyst-forming coccidia (Toxoplasma gondii, Neospora caninum, Sarcocystis spp.) in Mediterranean periurban micromammals.

Rodents and other micromammals constitute important reservoirs of infectious diseases; their role in the life cycle of apicomplexan parasites such as Toxoplasma gondii, Neospora caninum, and Sarcocystis spp. still needs clarification. In the present study, we analyzed by PCR and Sanger sequencing methods the presence of specific parasite DNA within brain and heart tissues of 313 individuals of five synanthropic small mammal species (Apodemus sylvaticus, Mus spretus, M. musculus, Rattus rattus, and Crocidura russula) collected in Barcelona metropolitan area (NE Spain). In addition, PCR-RFLP and microsatellites were also used as tools for genotypic characterization of T. gondii and N. caninum, respectively. Specific DNA of T. gondii, N. caninum, and Sarcocystis spp. was detected in 0.3% (n = 1), 1.3% (n = 4), and 3.8% (n = 12) of the animals, respectively. No mixed infections were observed. Crocidura russula stood out as the main host for Sarcocystis spp. Toxoplasma gondii-specific DNA detected in a house rat was genetically characterized by PCR-RFLP, presenting type II and III alleles (SAG1 [II], SAG3 [II], GRA6 [II], c22-8 [III], Apico [III]). Also, unsuccessful DNA sequencing and microsatellite typing were attempted in N. caninum-positive samples, which suggested a lack of PCR specificity and open avenues to speculate the host competence of rodents for N. caninum. Likewise, Sarcocystis spp. identity was studied by alignment and phylogenetic analyses of cox1 and 28S rRNA sequences from the 14 positive samples. It resulted in at least three unknown organisms closely similar (95.7-100% cox1-sequence homology) to Sarcocystis pantherophisi from the Eastern rat snake (Pantherophis alleghaniensis) (KU891603), suggesting together with 28S rRNA sequences analyses, three Sarcocystis sp. with a life cycle conformed by rodents as intermediate host (IH) and snakes as definitive hosts (DH) infecting the periurban micromammals surveyed. Prevalence figures found in this first survey carried out in Spain agree with other international studies focused on periurban areas. Further surveys should be conducted in farms and their surroundings in order to unravel the role of wild micromammals in the epidemiology of such protozoan parasites affecting our livestock, and therefore human population.

Symbiotic microbes and potential pathogens in the intestine of dead southern right whale (Eubalaena australis) calves.

Between 2003 and 2017, at least 706 southern right whale (Eubalaena australis) calves died at the Península Valdés calving ground in Argentina. Pathogenic microbes are often suggested to be the cause of stranding events in cetaceans; however, to date there is no evidence supporting bacterial infections as a leading cause of right whale calf deaths in Argentina. We used high-throughput sequencing and culture methods to characterize the bacterial communities and to detect potential pathogens from the intestine of stranded calves. We analyzed small and large intestinal contents from 44 dead calves that stranded at Península Valdés from 2005 to 2010 and found 108 bacterial genera, most identified as Firmicutes or Bacteroidetes, and 9 genera that have been previously implicated in diseases of marine mammals. Only one operational taxonomic unit was present in all samples and identified as Clostridium perfringens type A. PCR results showed that all C. perfringens isolates (n = 38) were positive for alpha, 50% for beta 2 (n = 19) and 47% for enterotoxin (CPE) genes (n = 18). The latter is associated with food-poisoning and gastrointestinal diseases in humans and possibly other animals. The prevalence of the cpe gene found in the Valdés' calves is unusually high compared with other mammals. However, insufficient histologic evidence of gastrointestinal inflammation or necrosis (the latter possibly masked by autolysis) in the gut of stranded calves, and absence of enterotoxin detection precludes conclusions about the role of C. perfringens in calf deaths. Further work is required to determine whether C. perfringens or other pathogens detected in this study are causative agents of calf deaths at Península Valdés.

Post-mortem findings in southern right whales Eubalaena australis at Península Valdés, Argentina, 2003-2012.

Between 2003 and 2012, 605 southern right whales (SRW; Eubalaena australis) were found dead along the shores of Península Valdés (PV), Argentina. These deaths included alarmingly high annual losses between 2007 and 2012, a peak number of deaths (116) in 2012, and a significant number of deaths across years in calves-of-the-year (544 of 605 [89.9%]; average = 60.4 yr(-1)). Post-mortem examination and pathogen testing were performed on 212 whales; 208 (98.1%) were calves-of-the-year and 48.0% of these were newborns or neonates. A known or probable cause of death was established in only a small number (6.6%) of cases. These included ship strike in a juvenile and blunt trauma or lacerations (n = 5), pneumonia (n = 4), myocarditis (n = 2), meningitis (n = 1), or myocarditis and meningitis (n = 1) in calves. Ante-mortem gull parasitism was the most common gross finding. It was associated with systemic disease in a single 1-2 mo old calf. Immunohistochemical labeling for canine distemper virus, Toxoplasma gondii and Brucella spp., and PCR for cetacean morbillivirus (CeMV), influenza A, and apicomplexan protozoa were negative on formalin-fixed, paraffin-embedded lung and brain samples from a subset of whales; PCR for Brucella spp. was positive in a newborn/neonate with pneumonia. Skin samples from whales with gull parasitism were PCR negative for CeMV, poxvirus, and papillomavirus. This is the first long-term study to investigate and summarize notable post-mortem findings in the PV SRW population. Consistent, significant findings within or between years to explain the majority of deaths and those in high-mortality years remain to be identified.

Differences in the ectoparasite fauna between micromammals captured in natural and adjacent residential areas are better explained by sex and season than by type of habitat.

We compared the ectoparasite fauna in 608 micromammals (chiefly 472 wood mice Apodemus sylvaticus, 63 Algerian mice Mus spretus, and 51 greater white-toothed shrews Crocidura russula) captured in natural and adjacent residential areas in spring and autumn during three consecutive years in four areas in periurban Barcelona (NE Spain). We found little support for an association of urbanization with differences in infestation by ectoparasites. Prevalence of Rhipicephalus sp. tick in wood mice and shrews was significantly higher in residential than in natural habitats, and the opposite was found for the flea Ctenophtalmus andorrensis catalanensis in shrews. Marked differences in the prevalence of the flea Leptopsylla taschenbergi amitina in wood mice between seasons were observed in natural but not in residential habitats, probably due to enhanced flea survival probabilities in the latter. However, as a rule, males were more frequently and heavily infested than females, and the prevalence was higher in autumn than in spring. Our results suggest that the ectoparasite fauna of periurban micromammals is shaped more by other factors than by habitat modification. People living in residential areas are at risk of contact with the arthropods borne by non-commensal micromammals and the pathogens transmitted by them.

Factors associated with the prevalence and pathology of Calodium hepaticum and C. splenaecum in periurban micromammals.

Calodium hepaticum (syn. Capillaria hepatica) and Calodium splenaecum (syn. Capillaria splenaecum) are nematodes that infect the liver and spleen, respectively, of mammals. While the host range, distribution, pathology and zoonotic potential of C. hepaticum are well known, very little is known about C. splenaecum. The observed prevalence of these two parasites, the factors associated with prevalence, and the lesions resulting in the different host species were studied in 408 micromammals captured in two periurban areas of Barcelona (NE Spain) from 2011 to 2013. C. hepaticum was found in 4% of 322 wood mice (Apodemus sylvaticus) (with local prevalence up to 16%) and 1 of 2 Norwegian rats (Rattus norvegicus). C. splenaecum was found in 10 % of 38 greater white-toothed shrew (Crocidura russula) (local prevalence up to 30%). Neither parasite was detected in 29 Algerian mice (Mus spretus) and 17 black rats (Rattus rattus). Prevalence of C. hepaticum was significantly higher in wood mice captured in natural areas (6.4%) than those from residential areas (0%), and infected mice were in better body condition. No differences in prevalence were found among age and sex groups, years and seasons. Lesions of hepatic capillariasis in wood mice consisted mainly of mild to moderate multifocal granulomas around degenerating adult parasites and/or eggs, while lesions seen in a rat consisted of multifocal granulomatous hepatitis and bridging fibrosis extending from the necrotic areas caused by the parasites. Splenic lesions found in shrews due to C. splenaecum, representing the first histological description of this parasite, were single nodules that corresponded to finely encapsulated clusters of eggs with adult parasites.

Anesthesia of Tibetan yak (Bos grunniens) using thiafentanil - xylazine and carfentanil - xylazine.

The use of 0.025 +/- 0.012 mg/kg (median +/- interquartile range) thiafentanil with 0.15 +/- 0.03 mg/kg xylazine (TX) and 0.011 +/- 0.0015 mg/kg carfentanil with 0.25 +/- 0.093 mg/kg xylazine (CX), with dosages based on estimated bodyweight, was used in the anesthesia of 37 Tibetan yak (Bos grunniens) housed within a drive-through animal park setting. The median time to lateral recumbency was 5 and 7 min for each group, respectively. With the addition of propofol in 8 CX animals and 17 TX animals, the anesthetic plane was suitable for a wide range of procedures. The median time to standing recovery following administration of naltrexone was 4 +/- 3.5 min with TX and 7 +/- 1.5 min with CX. There was one fatality and one case of renarcotization in the TX group. Overall, the dosages used in the study provided a reliable and useful anesthetic induction protocol, with TX animals demonstrating a more rapid induction and recovery with less cardiac depression than CX animals.

Effective Field Immobilization of Andean Fox (Lycalopex culpaeus) with Ketamine-Dexmedetomidine and Antagonism with Atipamezole.

A combination (mean±SD) of ketamine (4.0±1.0 mg/kg in juveniles and 3.0±0.4 in adults) and dexmedetomidine (0.055±0.01 and 0.049±0.01, respectively), reversed with atipamezole (at 10 mg/mg of dexmedetomidine), was assessed in 57 Andean foxes (Lycalopex culpaeus) in field conditions. Induction times in juveniles and adults were 4.6±3.9 min and 4.3±2.4 min, respectively. Immobilization was smooth and safe, and lasted 50±8 min in juveniles and 50±10 min in adults. Full recovery was recorded at 40±29 min in juveniles and 37±23 min in adults after atipamezole administration. Drug dose, season, body temperature, and fox sex and body condition were not related to variations in induction and recovery times, body temperature, heart rate, respiratory rate, or hemoglobin oxygen saturation. No side effects were observed other than a slight but significant decrease in mean body temperature during the procedure. This combination allowed carrying out all the typical procedures of a research project, including the collection of several biologic samples.

Drivers of Bartonella infection in micromammals and their fleas in a Mediterranean peri-urban area.

People living at the human/wildlife interface are at risk of becoming infected with Bartonella for which micromammals act as reservoir. We aimed to determine the factors related to the prevalence of Bartonella and its haplotype diversity in micromammals and in their fleas in a Mediterranean peri-urban environment. We analyzed 511 micromammals, chiefly 407 wood mice (Apodemus sylvaticus), captured into Barcelona metropolitan area (Spain) in spring and autumn from 2011 to 2013 in two natural and two adjacent residential areas, their fleas (grouped in 218 monospecific pools) and 29 fetuses from six Bartonella-positive female wood mice. Amplification of a fragment of ITS was carried out by real time PCR. Prevalence was 49% (57% in the dominant species, the wood mouse), and 12 haplotypes were detected. In general, prevalence was higher in those hosts more heavily infested by fleas, coincident with higher rates of capture, in autumn than in spring, and in adults than in juveniles. Prevalence did not differ between natural and residential areas except for one prevalent haplotype, which was more frequent in natural areas. Prevalence in flea pools (58%) was only explained by Bartonella occurrence in the pool host. In 56.4% of the flea pools with identified Bartonella haplotypes, we found the same haplotype in the host and in its flea pool. Prevalence in wood mouse fetuses was 69%, with at least one infected fetus in all litters, and two litters with all the fetuses infected. indicating that vertical transmission might be important in Bartonella epidemiology in the wood mouse. There is a hazard of Bartonella infection for people living in residential areas and those visiting peri-urban natural areas in Barcelona.

High Prevalence of Rickettsia spp. in Dog Fleas (Siphonaptera: Pulicidae) in Rural Uganda.

Fleas are known vectors of zoonotic agents. Thirty-five fleas, including 28 Ctenocephalides felis (Bouché), four Pulex irritans (L.), and three Echidnophaga gallinacea (Westwood) from 19 rural dogs from southwestern Uganda were analyzed for the presence of Rickettsia spp. (ompB, gltA, and 17 kDa fragment genes) and Bartonella spp. (rpoB and ITS genes) by PCR. Rickettsial DNA was detected in 27 out of 28 of Ct. felis and in two out of four P. irritans. None of the E. gallinacea specimens harbored Rickettsia DNA. Rickettsia felis was confirmed in 12 Ct. felis and in the two P. irritans specimens with positive PCR-results. In addition, the presence of Candidatus Rickettsia asemboensis was evidenced in 15 Ct. felis. Bartonella spp. was not amplified in any sample. Our survey indicates that R. felis, the agent of the flea-borne spotted fever, is present in the study area. Besides, this is the first description of Ca. R. asemboensis in Uganda.

Cellular and ultrastructural characterization of the grey-morph phenotype in southern right whales (Eubalaena australis).

Southern right whales (SRWs, Eubalena australis) are polymorphic for an X-linked pigmentation pattern known as grey morphism. Most SRWs have completely black skin with white patches on their bellies and occasionally on their backs; these patches remain white as the whale ages. Grey morphs (previously referred to as partial albinos) appear mostly white at birth, with a splattering of rounded black marks; but as the whales age, the white skin gradually changes to a brownish grey color. The cellular and developmental bases of grey morphism are not understood. Here we describe cellular and ultrastructural features of grey-morph skin in relation to that of normal, wild-type skin. Melanocytes were identified histologically and counted, and melanosomes were measured using transmission electron microscopy. Grey-morph skin had fewer melanocytes when compared to wild-type skin, suggesting reduced melanocyte survival, migration, or proliferation in these whales. Grey-morph melanocytes had smaller melanosomes relative to wild-type skin, normal transport of melanosomes to surrounding keratinocytes, and normal localization of melanin granules above the keratinocyte nuclei. These findings indicate that SRW grey-morph pigmentation patterns are caused by reduced numbers of melanocytes in the skin, as well as by reduced amounts of melanin production and/or reduced sizes of mature melanosomes. Grey morphism is distinct from piebaldism and albinism found in other species, which are genetic pigmentation conditions resulting from the local absence of melanocytes, or the inability to synthesize melanin, respectively.

Molecular detection of vector-borne pathogens in wild and domestic carnivores and their ticks at the human-wildlife interface.

Urbanization of natural areas is considered one of the causes of the current apparent emergence of infectious diseases. Carnivores are among the species that adapt well to urban and periurban environments, facilitating cross-species disease transmission with domestic dogs and cats, and potentially with their owners. The prevalence of vector-borne pathogens (VBP) of zoonotic and veterinary interest was studied in sympatric wild and domestic carnivores into Barcelona Metropolitan Area (NE Spain). Blood or spleen samples from 130 animals, including 34 common genets (Genetta genetta), 12 red foxes (Vulpes vulpes), 10 stone martens (Martes foina), three Eurasian badgers (Meles meles), 34 free-roaming domestic cats and 37 dogs with outdoor access, were collected either in protected or adjacent residential areas. A total of 309 ticks (chiefly Rhipicephalus turanicus) were collected on these animals. The samples were analyzed with a battery of PCR assays targeting the DNA of Rickettsia spp., Anaplasmataceae, Coxiella burnetii, Bartonella spp., and Piroplasmida, and the amplicons were sequenced. The fox showed the highest prevalence (58%) and diversity of VBP (four pathogens), whereas none of the dogs were infected. Bartonella spp. (including B. clarridgeiae, B. henselae, and B. rochalimae) was the most prevalent pathogen. Infection of wild carnivores with Ehrlichia canis, C. burnetii, Theileria annae and Babesia vogeli was also confirmed, with some cases of coinfection observed. The presence of DNA of T. annae and B. vogeli was also confirmed in tick pools from four species of wild carnivores, supporting their role in piroplasmid life-cycle. By the sequencing of several target genes, DNA of Rickettsia massiliae was confirmed in 17 pools of Rh. turanicus, Rh. sanguineous, and Rh. pusillus from five different species, and Rickettsia conorii in one pool of Rh. sanguineous from a dog. None of the hosts from which these ticks were collected was infected by Rickettsia. Although carnivores may not be reservoir hosts for zoonotic Rickettsia, they can have an important role as mechanical dispersers of infected ticks.

Serum chemistry reference values for the common genet (Genetta genetta): variations associated with Leishmania infantum infection.

BACKGROUND: The role of wildlife in the epidemiology of leishmaniosis in under debate, and determining whether infection with Leishmania infantum causes illness in wild carnivores is important to determine its potential role as a reservoir. OBJECTIVES: To provide for the first time serum biochemistry reference values for the common genet (Genetta genetta), and to determine variations associated with L. infantum infection. METHODS: Twenty-five serum biochemistry parameters were determined in 22 wild-caught genets. Blood samples were analyzed for L. infantum DNA by means of real-time polymerase chain reaction (PCR). RESULTS: Two female genets were positive for L. infantum DNA but did not show any external clinical sign upon physical examination. Among other variations in the biochemistry values of these genets, one presented a higher concentration of gamma-globulins and cholesterol, whereas the other genet presented increased creatinine, bilirubin, and chloride levels when compared to uninfected females. Sex-related differences in some parameters were also reported. CONCLUSION: Infection with L. infantum may sometimes be accompanied by abnormal serum biochemistry in wild carnivores. CLINICAL IMPORTANCE: Clinical disease may occur in L. infantum-infected wild carnivores. This has implications in the epidemiology of leishmaniosis. In addition, the data provided here would also be useful as reference values for researchers or rehabilitators working with the common genet.

Increased Wounding of Southern Right Whale (Eubalaena australis) Calves by Kelp Gulls (Larus dominicanus) at Península Valdés, Argentina.

At least 626 southern right whale (Eubalaena australis) calves died at the Península Valdés calving ground, Argentina, between 2003 and 2014. Intense gull harassment may have contributed to these deaths. In the 1970s, Kelp Gulls (Larus dominicanus) began feeding on skin and blubber pecked from the backs of living right whales at Valdés. The frequency of gull attacks has increased dramatically over the last three decades and mother-calf pairs are the primary targets. Pairs attacked by gulls spend less time nursing, resting and playing than pairs not under attack. In successive attacks, gulls open new lesions on the whales' backs or enlarge preexisting ones. Increased wounding could potentially lead to dehydration, impaired thermoregulation, and energy loss to wound healing. The presence, number and total area of gull-inflicted lesions were assessed using aerial survey photographs of living mother-calf pairs in 1974-2011 (n = 2680) and stranding photographs of dead calves (n = 192) in 2003-2011. The percentage of living mothers and calves with gull lesions increased from an average of 2% in the 1970s to 99% in the 2000s. In the 1980s and 1990s, mothers and calves had roughly equal numbers of lesions (one to five), but by the 2000s, calves had more lesions (nine or more) covering a greater area of their backs compared to their mothers. Living mother-calf pairs and dead calves in Golfo Nuevo had more lesions than those in Golfo San José in the 2000s. The number and area of lesions increased with calf age during the calving season. Intensified Kelp Gull harassment at Península Valdés could be compromising calf health and thereby contributing to the high average rate of calf mortality observed in recent years, but it cannot explain the large year-to-year variance in calf deaths since 2000.

Infection and exposure to vector-borne pathogens in rural dogs and their ticks, Uganda.

BACKGROUND: In rural parts of Africa, dogs live in close association with humans and livestock, roam freely, and usually do not receive prophylactic measures. Thus, they are a source of infectious disease for humans and for wildlife such as protected carnivores. In 2011, an epidemiological study was carried out around three conservation areas in Uganda to detect the presence and determine the prevalence of vector-borne pathogens in rural dogs and associated ticks to evaluate the risk that these pathogens pose to humans and wildlife. METHODS: Serum samples (n = 105), blood smears (n = 43) and blood preserved on FTA cards (n = 38) and ticks (58 monospecific pools of Haemaphysalis leachi and Rhipicephalus praetextatus including 312 ticks from 52 dogs) were collected from dogs. Dog sera were tested by indirect immunofluorescence to detect the presence of antibodies against Rickettsia conorii and Ehrlichia canis. Antibodies against R. conorii were also examined by indirect enzyme immunoassay. Real time PCR for the detection of Rickettsia spp., Anaplasmataceae, Bartonella spp. and Babesia spp. was performed in DNA extracted from FTA cards and ticks. RESULTS: 99% of the dogs were seropositive to Rickettsia spp. and 29.5% to Ehrlichia spp. Molecular analyses revealed that 7.8% of the blood samples were infected with Babesia rossi, and all were negative for Rickettsia spp. and Ehrlichia spp. Ticks were infected with Rickettsia sp. (18.9%), including R. conorii and R. massiliae; Ehrlichia sp. (18.9%), including E. chaffeensis and Anaplasma platys; and B. rossi (1.7%). Bartonella spp. was not detected in any of the blood or tick samples. CONCLUSIONS: This study confirms the presence of previously undetected vector-borne pathogens of humans and animals in East Africa. We recommend that dog owners in rural Uganda be advised to protect their animals against ectoparasites to prevent the transmission of pathogens to humans and wildlife.

Field immobilization of wood mice (Apodemus sylvaticus) with medetomidine and ketamine and antagonism with atipamezole.

Two anesthesia protocols using ketamine plus medetomidine and reversed with atipamezole were assessed for anesthesia of 148 wood mice (Apodemus sylvaticus) sampled under field conditions in northeastern Spain. In both protocols, induction was rapid (mean 76 sec), but recovery was slow (100 min), and mortality was relatively high (9%).