Barbara C. Shock

Natural history of Zoonotic Babesia: Role of wildlife reservoirs.

Graphical abstract Highlights ► All zoonotic Babesia utilize mammals, primarily wildlife, as reservoirs. ► Reservoirs and/or ticks are unknown for many zoonotic Babesia species. ► Molecular characterization of Babesia from human patients is needed. ► Continued surveillance of potential reservoirs and ticks should be conducted.

Distribution and prevalence of Cytauxzoon felis in bobcats (Lynx rufus), the natural reservoir, and other wild felids in thirteen states.

Cytauxzoon felis, a protozoan parasite of wild and domestic felids, is the causative agent of cytauxzoonosis in domestic and some exotic felids in the United States. The bobcat (Lynx rufus) is the natural reservoir for this parasite, but other felids such as Florida panthers (Puma concolor coryii) and domestic cats may maintain long-term parasitemias and serve as reservoirs. Experimentally, two tick species, Dermacentor variabilis and Amblyomma americanum, have demonstrated the ability to transmit C. felis. These two tick species have overlapping distributions throughout much of the southeastern United States. The objective of the current study was to determine the distribution and prevalence of C. felis in free-ranging bobcat populations from 13 states including California, Colorado, Florida, Georgia, Kansas, Kentucky, Missouri, North Carolina, North Dakota, Ohio, Oklahoma, South Carolina, and West Virginia. These states were selected because of differential vector presence; D. variabilis is present in each of these states except for the region of Colorado sampled and A. americanum is currently known to be present only in a subset of these states. Blood or spleen samples from 696 bobcats were tested for C. felis infection by a polymerase chain reaction (PCR) assay which targeted the first ribosomal internal transcribed spacer region (ITS-1). Significantly higher prevalences of C. felis were detected from Missouri (79%, n=39), North Carolina (63%, n=8), Oklahoma (60%, n=20), South Carolina (57%, n=7), Kentucky (55%, n=74), Florida (44%, n=45), and Kansas (27%, n=41) compared with Georgia (9%, n=159), North Dakota (2.4%, n=124), Ohio (0%, n=19), West Virginia (0%, n=37), California (0%, n=26), and Colorado (0%, n=67). In addition to bobcats, seven cougars (Puma concolor) from Georgia, Louisiana, and North Dakota and one serval (Leptailurus serval) from Louisiana were tested for C. felis. Only one cougar from Louisiana was PCR positive, which represents the first report of an infected cougar outside of the Florida panther population. These data also indicate that C. felis is present in North Dakota where infection has not been reported in domestic cats. Based on a nonparametric analysis, prevalence rates were significantly higher in states where there are established populations of A. americanum, which supports recent data on the experimental transmission of C. felis by A. americanum and the fact that domestic cat clinical cases are temporally associated with A. americanum activity. Collectively, these data confirm that bobcats are a common reservoir for C. felis and that A. americanum is likely an epidemiologically important vector.

Novel relapsing fever Borrelia detected in African penguins ( Spheniscus demersus ) admitted to two rehabilitation centers in South Africa

The African penguin, Spheniscus demersus, the only penguin species that breeds in Africa, is endangered, and several diseases including avian malaria, babesiosis, and aspergillosis are common in some populations. From 2002 to 2010, spirochetes morphologically consistent with Borrelia were observed on thin blood smears from 115 of 8,343 (1.4%) African penguins admitted to rehabilitation centers in the Western Cape and Eastern Cape provinces of South Africa. Prevalence rates were significantly higher among chicks and juveniles compared with adults and for birds sampled during the summer months of October to February compared with winter months. The majority of infected birds were ultimately released, despite lack of antibiotic treatment; however, at least one bird is believed to have died of borreliosis based on characteristic gross and microscopic lesions. Analysis of partial flaB gene sequences indicated this was a relapsing fever Borrelia most similar to a Borrelia sp. detected in soft ticks from a seabird colony in Japan. This represents the fourth report of a relapsing fever Borrelia sp. in an avian species and highlights the need for additional studies of potentially pathogenic organisms infecting the African penguin in South Africa.

Diversity of piroplasms detected in blood-fed and questing ticks from several states in the United States.

Piroplasms in the genera Babesia, Theileria, and Cytauxzoon are tick-borne parasites that may be animal and human pathogens. Most piroplasms with known life cycles are transmitted by ixodid ticks; however, for many species, the vector is unknown. This study was conducted to determine the prevalence and diversity of piroplasms in ticks from several US states. Piroplasm-specific polymerase chain reaction (PCR) assays were used to test 1631 ticks from Georgia (n=486), Kentucky (n=103), Pennsylvania (n=1), Tennessee (n=626), and Texas (n=414). Ticks were either questing (n=42) or collected from animals (n=627) or humans (n=962). The 2 primary species tested were Dermacentor variabilis (n=702) and Amblyomma americanum (n=743), but Amblyomma cajennense (n=99), Amblyomma maculatum (n=16), Ixodes scapularis (n=4), I. woodi (n=1), and unidentified Amblyomma spp. nymphs (n=64) were also tested. A low prevalence of piroplasms was detected with 37 (2.3%), 35 (2.1%), and 9 (0.6%) ticks positive for Theileria spp., Babesia spp., or Cytauxzoon felis, respectively. Based on sequence analysis, at least 6 Babesia spp. were detected and 15 of the 35 (41%) Babesia-positive ticks were A. americanum, 19 (56%) were D. variabilis, and one (3%) was an I. scapularis. Nine Babesia-positive ticks were removed from humans from Kentucky (n=1), Georgia (n=2), Texas (n=5), and Pennsylvania (n=1). Three Babesia-positive ticks were questing A. americanum which represents the first report of Babesia-infected questing Amblyomma in the US. Theileria infections were only detected in A. americanum, and all sequences were similar to white-tailed deer associated Theileria spp. C. felis was only detected in D. variabilis. These data suggest that A. americanum may be a vector of Babesia spp., although experimental studies are needed to confirm vector competence. Finally, these data demonstrate a high diversity of piroplasms in both questing and partially fed ticks in the US; although, host-blood meals can be present in non-questing ticks.

Variation in the ITS-1 and ITS-2 rRNA genomic regions of Cytauxzoon felis from bobcats and pumas in the eastern United States and comparison with sequences from domestic cats.

Cytauxzoon felis, a tick-borne protozoan parasite, is the causative agent of cytauxzoonosis in domestic cats in the United States. The natural reservoir for this parasite is the bobcat (Lynx rufus), which typically does not develop clinical signs. Although not likely important reservoirs, C. felis has also been detected in pumas (Puma concolor) in Florida and Louisiana. Recent studies suggest that specific genotypes of C. felis that circulate in domestic cats may be associated with variable clinical outcomes and specific spatial locations. In the current study, we investigated the intraspecific variation of the C. felis internal transcribed spacer (ITS)-1 and ITS-2 rRNA regions from 145 wild felids (139 bobcats and six pumas) from 11 states (Florida, Georgia, Kansas, Kentucky, Louisiana, Missouri, North Carolina, North Dakota, South Carolina, Oklahoma, and Pennsylvania). Unambiguous ITS-1 and ITS-2 data were obtained for 144 and 112 samples, respectively, and both ITS-1 and ITS-2 sequences were obtained for 111 (77%) samples. For the ITS-1 region, sequences from 65 samples collected from wild felids were identical to those previously reported in domestic cats, while the other 79 sequences were unique. C. felis from 45 bobcats and one puma had ITS-1 sequences identical to the most common sequence reported from domestic cats. Within the ITS-2 region, sequences from 49 bobcats were identical to those previously reported in domestic cats and 63 sequences were unique (with some occurring in more than one bobcat). The most common ITS-2 sequence from domestic cats was also common in wild felids (31 bobcats and a puma). Samples from three pumas from Florida and two bobcats from Missouri had a 40- or 41-bp insert in the ITS-2 similar to one described previously in a domestic cat from Arkansas. Additionally, a previously undescribed 198- or 199-bp insert was detected in the ITS-2 sequence from four bobcats. Collectively, based on combined ITS-1 and ITS-2 sequences, five different genotypes were detected in the wild felids. Genotype ITSa was the most common genotype (11 bobcats and one puma) and fewer numbers of ITSb, ITSe, ITSg, and ITSi were detected in bobcats. These data indicate that, based on ITS-1 and ITS-2 sequences, numerous C. felis strains may circulate in wild felids.

Effects of Salinity Stress on Survival, Metabolism, Limb Regeneration, and Ecdysis in UCA pugnax

Abstract Estuarine intertidal zones are characterized by variable salinity regimes due to seasonal and daily fluctuations. Salt-marsh inhabitants, such as the mud fiddler crab Uca pugnax, are physiologically adapted to this habitat. Crustacean growth and development are energy demanding and sensitive hormonal processes which are susceptible to natural stressors. This study investigated physiological and metabolic changes in the molt cycle of U. pugnax in response to different salinity levels by assessing the survival, respiration, and post-molt tissue protein and carbohydrate content. Crabs were subjected to a limb regeneration and ecdysis challenge under four different salinity regimes. For the study, a limb was removed and its regenerative growth was photographed every two days. After ecdysis, crabs were dissected, and the tissues collected were analyzed for their protein and carbohydrate contents. Crabs held at 60 and 75 ppt seawater showed 100% mortality. Crabs at 60 ppt did not undergo basal limb growth. During limb regeneration, abnormal limb growth occurred solely in males. Post-molt 40 ppt crabs showed elevated respiration rates compared to 10 ppt crabs. Females and 25 ppt crabs had significantly more free carbohydrates in epithelial tissue than males and crabs at 10 and 40 ppt. As salinity decreased, epithelial protein content in females decreased while males experienced no change. Salinity did not affect the amount of acid, water or base-soluble exoskeleton protein. These physiological responses indicate that U. pugnax males and individuals exposed to extreme salinities are more likely to be impacted by salinity stress during limb regeneration and ecdysis.

Parasites and vector-borne pathogens of southern plains woodrats (Neotoma micropus) from southern Texas

From 2008 to 2010, southern plains woodrats (Neotoma micropus) from southern Texas, were examined for parasites and selected pathogens. Eight helminth species were recovered from 97 woodrats including, Trichuris neotomae from 78 (prevalence = 80%), Ascarops sp. from 42 (43%), Nematodirus neotoma from 31 (32%), Raillietina sp. from nine (9%), Taenia taeniaeformis larvae from eight (8%), and an unidentified spiurid, a Scaphiostomum sp. and a Zonorchis sp. each from a single woodrat. Besnotia neotomofelis was detected in three (3%) woodrats and microfilaria were detected in seven (7%). Polymerase chain reaction (PCR) testing of blood samples from 104 woodrats detected a novel Babesia sp. in one (1%) and Hepatozoon sp. in 17 (16%) woodrats. Partial 18S rRNA gene sequence of the Babesia was 94% similar to B. conradae. Histologic examination of tissues detected intestinal coccidia in seven of 104 (7%), Sarcocystis neotomafelis in 26 (25%), Hepatozoon sp. in 21 (20%), and Dunnifilaria meningica in four (4%) woodrats. Three woodrats (5%) were seropositive for Toxoplasma gondii. Ectoparasites recovered included fleas (Orchopeas sexdentatus and O. neotomae), ticks (Ixodes woodi and Ornithodoros turicata), mites (Trombicula sp. and Ornithonyssus (Bdellonyssus) bacoti) and bot flies (Cuterebra sp.). The only difference in prevalence related to gender was for N. neotoma (males > females, p = 0.029). Prevalence of T. neotomae and all intestinal parasites combined was significantly higher in adults compared with juveniles (p = 0.0068 and p = 0.0004), respectively. Lesions or clinical signs were associated with Cuterebra and B. neotomofelis. Collectively, these data indicate that woodrats from southern Texas harbor several parasites of veterinary and/or medical importance.

White-Nosed Coatis (Nasua narica) Are a Potential Reservoir of Trypanosoma cruzi and Other Potentially Zoonotic Pathogens in Monteverde, Costa Rica

Abstract We studied white-nosed coatis (Nasua narica) in Monteverde, Costa Rica to evaluate their potential as a reservoir for the vector-borne zoonotic parasite Trypanosoma cruzi and other selected microorganisms. We live-trapped 20 coatis in July and August 2011 and tested them for T. cruzi by blood smear, molecular analysis of blood, culture of blood and anal gland secretions, and serology. Seven coatis (35%) were polymerase-chain-reaction–positive for T. cruzi and one coati was also culture positive. We did not detect T. cruzi in anal gland secretions. All coatis were positive for Mycoplasma and Babesia, but were negative for Baylisascaris, Anaplasma, Candidatus Neoehrlichia lotoris, Ehrlichia, Bartonella, and several apicomplexan parasites. The possible pathogenicity of T. cruzi, Babesia, and Mycoplasma in coatis and their transmission potential to humans and domestic animals warrants further investigation.

Multiple stressor effects of methoprene, permethrin, and salinity on limb regeneration and molting in the mud fiddler crab (UCA pugnax)

Exposure to multiple stressors from natural and anthropogenic sources poses risk to sensitive crustacean growth and developmental processes. Applications of synthetic pyrethroids and insect growth regulators near shallow coastal waters may result in harmful mixture effects depending on the salinity regime. The potential for nonadditive effects of a permethrin (0.01 2 gg/L), methoprene (0.03-10 tg/L ), and salinity (10-40 ppt) exposure on male and female Uca pugnax limb regeneration and molting processes was evaluated by employing a central composite rotatable design with multifactorial regression. Crabs underwent single-limb autotomy followed by a molting challenge under I of 16 different mixture treatments. During the exposure (21-66 d), individual limb growth, major molt stage duration, abnormal limb regeneration, and respiration were monitored. At 6 d postmolt, changes in body mass, carapace width, and body condition factor were evaluated. Dorsal carapace tissue was collected, and protein and chitin were extracted to determine the composition of newly synthesized exoskeleton. The present results suggest chronic, low-dose exposures to multiple pesticide stressors cause less-than-additive effects on U. pugnax growth processes. Under increasing concentrations of methoprene and permethrin, males had more protein in their exoskeletons and less gain in body mass, carapace width, and body condition compared to females. Females exhibited less gain in carapace width than controls in response to methoprene and permethrin. Females also displayed elevated respiration rates at all stages of molt, suggesting a high metabolic rate. Divergent growth and fitness between the sexes over the long term could influence crustacean population resilience.

Fatal hepatic tetratrichomoniasis in a juvenile Waldrapp ibis (Geronticus eremita)

Waldrapp ibis (Geronticus eremita) are a critically endangered species, and there are currently more birds in captivity than in the wild. A juvenile, male Waldrapp ibis housed in a mixed-species exhibit was found dead with no premonitory signs. Necropsy revealed extensive necrotizing hepatitis associated with numerous pleomorphic protozoa that were immunohistochemically reactive with antibodies raised against Tritrichomonas foetus, a parasite of cattle. Electron microscopy confirmed the organisms as members of family Trichomonadidae, and sequence analysis of the first ribosomal internal transcribed spacer region (ITS1), 5.8S ribosomal RNA, and ITS2 regions indicated high genetic similarity (96–97%) to members of the Tetratrichomonas gallinarum complex. The animal was born in captivity, and no introductions in this exhibit had occurred since 2009. Other Waldrapp ibis that had contact with the infected male were negative for flagellate infections by fecal examination, thus cross-species transmission is proposed as the source of infection. The host range of the T. gallinarum complex is very large and although the pathogenicity of its members, especially for wild birds, is controversial, these parasites should be considered as a possible cause of acute mortality in Waldrapp ibis. In addition, immunohistochemistry with T. foetus antibodies and molecular diagnostics may be useful tools for preventative veterinary care of endangered bird populations. A greater understanding of the ecology and pathogenesis of this pathogen may also be vital for screening subclinical captive populations and existing wild populations prior to reintroduction efforts.