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Babesia spp. in Ursus americanus (Black Bear) in New Jersey
Melissa Shaw, Nikolai Kolba, and Jane E. Huffman

Northeastern Naturalist, Volume 22, Issue 3 (2015): 451–458

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Northeastern Naturalist Vol. 22, No. 3 M. Shaw1, N. Kolba1, and J.E. Huffman 2015 451 2015 NORTHEASTERN NATURALIST 22(3):451–458 Babesia spp. in Ursus americanus (Black Bear) in New Jersey Melissa Shaw1, Nikolai Kolba1, and Jane E. Huffman1,* Abstract - Babesia is emerging as a cause of tick-borne zoonoses worldwide, and various wildlife species are the principal reservoir hosts for zoonotic Babesia species. The primary vectors of Babesia are Ixodid ticks, with the majority of zoonotic species transmitted by species in the genus Ixodes. The protozoan infects and lyses red blood cells. In this study, blood was collected from 201 Ursus americanus (American Black Bear) from 5 counties in northwestern New Jersey. Sample collection occurred over 5 research-trapping seasons from March 2010 to August 2011. We screened blood samples for Babesia spp. by nested polymerase chain reaction (PCR), amplifying a 448–992-bp portion of the 18S rRNA gene, and detected Babesia in 84 of 201 (41.8%) samples. Sequence analysis confirmed the presence of Babesia spp. in all of the PCR positive samples. This study represents the first report of molecular evidence of Babesia spp. in the American Black Bear. Introduction Babesiosis, or tick fever, is a febrile disease of humans and domestic and wild animals that is characterized by extensive erythrocytic lysis leading to anemia, icterus, and hemoglobinuria. It can be fatal. More than 100 species of Babesia have been described in a number of vertebrate hosts (Chauvin et al. 2009). Babesia microti, which usually infects Peromyscus leucopus (Rafinesque) (White-footed Mouse) and other small mammals, is the most common causal agent in human babesiosis. Clark et al. (2012) reported on the occurrence of Babesia microti in rodents and Procyon lotor (L.) (Raccoon) from northeast Florida, and Hirata et al. (2013) detected Babesia sp. NV-1 from Neovison vison (Schreber) (American Mink), which had been imported to Japan in the 1920s. In the northeastern US, Babesia microti is transmitted by Ixodes scapularis Say (Blacklegged Tick) (Spielman et al. 1985). Stiles and Baker (1935) reported a haemosporidean parasite (Babesia sp.) in a blood smear collected from an unidentified bear in a zoo in St. Petersburg, FL. Using molecular techniques, Babesia has been recently described in the family Ursidae. Jinnai et al. (2010) provided molecular evidence of Babesia infection of a wild Ursus arctos yesoensis L. (Hokkaido Brown Bear). Ikawa et al. (2011) reported Babesia sp. from Ursus tibetanus G. (Baron) Cuvier (Japanese Black Bear). Any mammal that is exposed to Blacklegged Ticks infected with B. microti is commonly considered to be a potential reservoir for the protozoan (Telford et al. 1993). Ursus americanus Pallas (American Black Bear, hereafter Black Bear) is omnivorous, solitary in nature, long-lived, and has a restricted home range in New Jersey. When Black Bears were surveyed in northern New Jersey, Blacklegged Ticks were 1Northeast Wildlife DNA Laboratory, Department of Biological Sciences, East Stroudsburg University, East Stroudsburg, PA 18301. *Corresponding author - jhuffman@esu.edu. Manuscript Editor: Howard S. Ginsberg Northeastern Naturalist 452 M. Shaw1, N. Kolba1, and J.E. Huffman 2015 Vol. 22, No. 3 a common ectoparasite (Burguess and Huffman 2005). During a different study of Blacklegged Ticks collected from Black Bears from northern New Jersey, 8.6% (19/220) of the ticks screened were found to be positive for B. microti (Bove 2012). Adelson et al. (2004) reported on the prevalence of B. microti in Blacklegged Ticks from northern New Jersey (primarily Union County) and, using the polymerase chain reaction (PCR), identified Babesia microti in 8.4% (9/107) of ticks examined. Health surveys of Black Bears and other wildlife species can provide valuable information about the potential for exposure to infectious or parasitic agents (Buttke et al. 2015, Dantes-Torres 2012, Stephen 2014, Yabsley and Shock 2013). The purpose of this study was to examine blood samples from New Jersey Black Bears for molecular evidence of Babesia spp. using PCR and sequence analysis. Methods New Jersey Division of Fish and Wildlife (NJDFW) biologists collected blood samples from 201 Black Bears in 5 northwestern counties in New Jersey (Warren, Sussex, Passaic, Morris, and Hunterdon) over 5 research-trapping seasons: March, June, and October 2010 and March and June 2011. NJDFW personnel established trap lines using Aldrich foot snares (checked every 24 hours) in northern New Jersey and ran the lines for 19 consecutive days during the trapping period. NJDFW personnel located the dens of radio- and satellite-collared sows in February and March 2010 and 2011 and collected blood samples from the sows and their cubs or yearlings at these sites. Black Bears were anesthetized with a combination of 200 mg/mL ketamine and 45 mg/mL xylazine administered via dart gun. Data collected for each animal included body measurements, weight, and sex. Biologists recorded ear-tag numbers and tattooed the right-ear tag number on the inside of the bear’s lip. The Black Bears were divided into 3 age classes: adults (>18 months), yearlings (12–18 months), and cubs (less than 12 months). NJDFW personnel collected blood samples from the femoral vein of juvenile and adult bears using a BD Vacutainer safety-lok Blood Collection set 21G x ¾” x 12” (BD, Franklin Lakes, NJ) and transferred each one into a 7-ml BD Vacutainer K3 containing EDTA. Biologists obtained blood samples from Black Bear cubs during den work at the time of ear tagging by collecting samples into 2-ml BD Vacutainer K3 containing EDTA. Blood samples were stored in a cooler in the field, delivered to the laboratory, processed within 12 h of collection, stored in 2-ml microcentrifuge tubes, and stored at -20 °C. We extracted DNA from 200-μl EDTA/whole blood samples with MO BIO UltraCleanTM BloodSpinTM Kit (MO BIO Laboratories, Carlsbad, CA) according to the manufacturer’s protocol. We used the Qubit Fluorometer (Invitrogen, Carlsbad, CA) to quantify extracted DNA following the manufacturer’s protocol. To detect Babesia, we conducted a PCR protocol that targeted the 18S rRNA gene (Pershing et al. 1995). For each PCR, we added 2.5 μl (0.1μg) of extracted DNA to 12.5 μL Promega Mastermix (GoTaq Colorless 2x; Promega Corporation, Madison, WI), 0.5 μL of each primer (50 μM), and 9 μl nuclease-free water, in a Northeastern Naturalist Vol. 22, No. 3 M. Shaw1, N. Kolba1, and J.E. Huffman 2015 453 total volume of 25 μl. The mixture for all secondary reactions was the same with the exception that we removed 1 μl of the resulting PCR product from the primary reaction to use as the template. The primary reaction was performed with primers 3.1: 5'-CTCCTTCCTTTAAGTGATAAG- 3' and 5.1: 5'-CCTGGTTGATCCTGCCAGTAGT-3' (Yabsley et al. 2005). The secondary reaction was performed using primers RLB-F: 5'-GAGGTAGTGACAAGAAATAACAATA- 3' and RLB-R: 5'-TCTTCGATCCCCTAACTTTC- 3' (Schouls et al. 1999). Primary PCR was carried out according to the following parameters: 94 oC for 3 min followed by 30 cycles of 94 oC for 1 min, 55 oC for 1 min, 72 oC for 1.5 min, and an extension step at 72 oC for 5 min. The secondary PCR was performed under the following conditions: 1 min at 94 oC followed by 40 cycles of 94 oC for 1 min, 50 oC for 1 min, 72 oC for 1.5 min, and a final extension step at 72 oC for 10 min. We included a positive control for Babesia sp. in each PCR and a negative water control in each set of primary and secondary PCRs. We electrophoresed the resulting PCR products on a 2%-agarose gel, stained the gel with ethidium bromide, and visualized it under UV light. PCR-positive products were purified of primer dimers and other nonspecific amplification by-products using ExoSAP-IT for PCR Product Clean-up (Affymetrix, Cleveland, OH) prior to sequencing. We sequenced the products using BigDye®Terminator v3.1 Cycle Sequencing Kits (Applied Biosystems, Foster City, CA) and ABI PRISM® 3130-Avant Genetic Analyzer (Applied Biosystems) and analyzed them with Sequencing Analysis ver. 5.2 (Applied Biosystems). We aligned sequences of 18S rRNA with those from related organisms obtained from Gen Bank using a basic alignment-search tool (BLAST; National Center for Biotechnology Information, Bethesda, MD) (Altschul et al. 1990). Sequence alignments were performed for all samples. We used the ClustalW (http://www.ch.embnet.org/software/ClustalW.html) program for sequence alignment. We obtained known Babesia spp. sequences from GenBank for sequence alignment and phylogenetic analysis and used Plasmodium falciparum as the out-group. We adjusted to corresponding equivalent lengths all sequences included in the alignment for phylogenetic analysis and used bootstrap analysis to assess reliability (1000 replicates). Samples that supported clades are shown on nodes for maximum parsimony analysis. We employed Dendroscope version 3.2.10 to view and edit the phylogenetic tree (Hudson and Scornavacca 2012). Results Positive PCR assays were characterized by banding present on the ethidium bromide- stained agarose gel at approximately 448–992 base pairs. The results for the positive and negative controls were correct for each assay performed. Eighty-four of 201 (41.8%) blood samples were PCR positive and sequence results confirmed Babesia spp. infection. We obtained PCR-positive samples from all 5 counties tested— Hunterdon (50.0%); Morris (47.8%); Passaic (37.0%); Sussex (38.2%), and Warren (48.6%). Babesia spp. was confirmed by PCR and sequence data in each of the age classes from which blood samples were collected. The adults and yearlings Northeastern Naturalist 454 M. Shaw1, N. Kolba1, and J.E. Huffman 2015 Vol. 22, No. 3 exhibited a greater rate of infection (44.1% and 42.6%, respectively) compared to cubs (20.0%). The prevalence rate of Babesia spp. was 46.1% and 38.0% in male and female Black Bears, respectively. Seasonal prevalence rates for 2010 and 2011 were 30.0% in March, 50.8% in June, and 27.0% in October. One sow and 1 of her cubs were positive by PCR for Babesia, and sequencing the respective amplicons confirmed that the sequences were identical. We obtained partial 18S rRNA gene sequences for all 84 PCR-positive samples. Resultant sequences shared the highest identity with 4 Babesia spp. in the database. Eleven samples matched most closely (93–100% match) with Babesia sp. MA#230 from feral Raccoons in Japan (AB251608). Sixteen samples matched closely (99–100%) with Babesia microti isolate P8803 (AY144701). Fifty-five samples matched closely (98–100%) with Babesia sp. AJB-2006 (DQ028958; Birkenheuer et al. 2007). One sample matched (99%) with Babesia coco (EU109716), a newly recognized Babesia sp. found in Canis lupus familiaris L. (Domestic Dog) in North Carolina. One sample matched (93%) a Babesia canis vogeli (EF052627) isolate from Domestic Dogs in Brazil (Table 1). A phylogenetic tree based on sequences of the 18S rRNA of Babesia spp. from the New Jersey Black Bears is shown in Figure 1. Discussion Babesia is a common infectious agent of free-living animals around the world (Homer et al. 2000). It has been shown that babesial DNA does not remain within the host very long after resolution of the parasitic infection (Krause et al. 1998). Babesia spp. have been reported at prevalences up to 96% within free-living animal populations (Frerichs and Holbrook 1970). The prevalence of B. microti within New Jersey Black Bears (38%) is consistent with other species of Babesia within other mammal populations (Sinski et al. 2006, Yabsley et al. 2006). This study is the first report of Babesia spp. in American Black Bears using 18S rRNA gene sequences for phylogenetic analysis. Babesia spp. has been reported in the family Ursidae, both from Japan (Ikawa et al. 2011, Jinnai et al. 2010) and from an unidentified bear in the US by Stiles and Baker (1935). In the current study, the prevalence rate of Babesia in adult and yearling Black Bears was significantly different than in the cubs. This result may be related to the age of the host. Adults and Table 1. The number of babesial samples sequenced with the resultant NCBI accession number, percent match, and the NCBI identification number. n = number of samples. NCBI n accession # % match NCBI identification number 11 AB251608 93–100 Babesia sp. MA#230 gene for 18S ribosomal RNA, partial sequence 16 AY144701 99–100 Babesia microti isolate P8803 18S ribosomal RNA gene, partial 55 DQ028958 98–100 Babesia sp. AJB-2006 18S ribosomal RNA gene, partial sequence 1 EU109716 99 Babesia sp. Coco 18S ribosomal RNA gene, partial sequence 1 EF052627.1 93 Babesia canis vogeli isolate RP5 18S ribosomal RNA gene, partial sequence Northeastern Naturalist Vol. 22, No. 3 M. Shaw1, N. Kolba1, and J.E. Huffman 2015 455 yearlings have more time to encounter ticks and a greater chance of being infected by the protozoan. Razi Jalali et al. (2013) reported that the infection rate was higher in adult Domestic Dogs 3–6 yr-old (4.46%, 5/112) compared with those less than 3-yr old (3.59%, 7/195). The most common route of Babesia infection is the bite of a competent vector tick. Transmission can also occur by transfusion of infected blood products, and vertical transmission in animals has been documented (de Vos et al. 1976, Fukumoto et al. 2005). A Babesia gibsoni-infected female Domestic Dog was mated with an uninfected male in order to determine whether this parasite could be vertically transmitted. The results showed that vertical transmission occurred by the uterine route and not via the transmammary route. This was the first confirmed report of transplacental Babesia infection in any animal species (Fukumoto et al. 2005). Joseph et al. (2012) reported a case of babesiosis in a 6-wk-old infant for whom vertical transmission was suggested by evidence of Babesia spp. antibodies in the heel-stick blood sample, and transplacental transmission was confirmed by detection of Babesia spp. DNA in placenta tissue. In the current study, 1 sow and 1 of her cubs was infected, and sequencing confirmed that the sequences were identical possibly indicating transplacental infection. The analyses performed on the babesial DNA from this study matched to either Raccoons or Domestic Dogs in the GenBank database. This finding may indicate that these babesial species are not as host-specific as once thought, particularly due Figure 1. Phylogenetic tree illustrating the position of Babesia isolates from NJ Black Bears to other isolates that have been reported. The isolates from Japanese bears Babesia sp. UR1 gene for 18S ribosomal RNA partial sequence (AY190124) and Babesia sp. Iwate248 gene for 18S ribosomal RNA (AB586027) are included in the tree. Specific genes within the tree that were close to isolates from Black Bears in this study are indicated by an asterisk. Northeastern Naturalist 456 M. Shaw1, N. Kolba1, and J.E. Huffman 2015 Vol. 22, No. 3 to the clear difference between these sequences. Our analyses placed another grouping of babesial isolates in the same clade as other B. microti-like species with high confidence. The other sequences were placed in the Babesia spp. sensu stricto clade with other species derived from Raccoons and Japanese Black Bears. However, some of the phylogenetic branches within the Babesia spp. sensu stricto clade show low bootstrap support. Similar findings have been observed in several other studies and are likely due to the fact that there are no genetic data available for many of the Babesia spp. in the sensu stricto clade (Holman et al. 2000, Zahler et al. 2000). 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