Publications by authors named "Kelly E Allen"

20 Publications

  • Page 1 of 1

Ticks infesting dogs and cats in North America: Biology, geographic distribution, and pathogen transmission.

Vet Parasitol 2021 Jun 19;294:109392. Epub 2021 Feb 19.

Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma, 74078, United States.

A diverse array of ixodid and argasid ticks infest dogs and cats in North America, resulting in skin lesions, blood loss, and disease. The ticks most commonly found on pets in this region are hard ticks of the genera Amblyomma, Dermacentor, Ixodes, and Rhipicephalus, as well as the more recently established Haemaphysalis longicornis. Soft tick genera, especially Otobius and Ornithodoros, are also reported from pets in some regions. In this review, we provide a summary of the complex and diverse life histories, distinct morphologies, and questing and feeding behaviors of the more common ticks of dogs and cats in North America with a focus on recent changes in geographic distribution. We also review pathogens of dogs and cats associated with the different tick species, some of which can cause serious, potentially fatal disease, and describe the zoonotic risk posed by ticks of pets. Understanding the natural history of ticks and the maintenance cycles responsible for providing an ongoing source of tick-borne infections is critical to effectively combatting the challenges ticks pose to the health of pets and people.
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http://dx.doi.org/10.1016/j.vetpar.2021.109392DOI Listing
June 2021

Detection of Cercopithifilaria bainae infection in shelter dogs and ticks in Oklahoma, USA.

Parasit Vectors 2020 Apr 25;13(1):216. Epub 2020 Apr 25.

Department of Veterinary Pathobiology, Oklahoma State University College of Veterinary Medicine, Stillwater, OK, USA.

Background: Cercopithifilaria bainae is a filarioid nematode of dogs. Infection with the parasite was not reported in the USA until 2017, when a dog with skin lesions in Florida was diagnosed. Brown dog ticks, Rhipicephalus sanguineus (sensu lato), are the purported tick vectors, and are widespread in the USA. Therefore, C. bainae is likely present in additional states. Here, we tested dogs and ticks in Oklahoma for evidence of C. bainae infection.

Methods: Dermal punch biopsies were opportunistically collected from municipal shelter and client-owned dogs. Multiple skin samples collected from interscapular and head regions were tested by saline sedimentation to recover live microfilariae for morphometric identification and by PCR to amplify a 330 bp region of the filarioid 12S rRNA gene. Also, ticks observed on surveyed dogs were collected, identified to species level, and tested for filarioid DNA.

Results: A total of 496 saline sedimentations were performed on 230 shelter and 20 client-owned dogs. Cercopithifilaria bainae infections were identified in 2.6% (6/230) of shelter dogs by morphometry of microfilariae in sedimentations and/or amplification of DNA from skin. DNA sequences amplified from PCR positive skin samples were 99-100% identical to C. bainae reported in Italy. All skin samples from client-owned dogs were negative for filarioid infection by saline sedimentation and PCR. A total of 112 ticks, comprised of four species, were collected. Two of 72 R. sanguineus (s.l.), both engorged females found attached to a C. bainae infected dog, harbored C. bainae DNA (99-100% identity). One attached R. sanguineus (s.l.) male on the same dog harbored filarioid DNA sequence which was difficult to interpret at numerous base-pair locations, but was closest in identity (~80%) to C. bainae.

Conclusions: The distribution of C. bainae is more widespread than previously known. To our knowledge, we document C. bainae infections in dogs and DNA in brown dog ticks in Oklahoma for the first time. As brown dog ticks are commonly found throughout the USA, veterinarians in this region should consider C. bainae infection as a differential diagnosis in canine patients with dermatitis or polyarthritis.
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http://dx.doi.org/10.1186/s13071-020-04089-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7183667PMC
April 2020

Transmission of Cytauxzoon felis to domestic cats by Amblyomma americanum nymphs.

Parasit Vectors 2019 Jan 11;12(1):28. Epub 2019 Jan 11.

Department of Veterinary Pathobiology, Oklahoma State University Center for Veterinary Health Sciences, Stillwater, OK, 74078, USA.

Background: Successful Cytauxzoon felis transmission studies have occurred using Amblyomma americanum adults acquisition-fed as nymphs on an experimentally infected domestic cat or Dermacentor variabilis adults fed as nymphs on a splenectomized bobcat. Here, we evaluated A. americanum and D. variabilis nymphs acquisition-fed as larvae on a C. felis-infected carrier domestic cat for competence to transmit the protozoan parasite as nymphs to naïve, healthy domestic cats.

Methods: Amblyomma americanum and D. variabilis larvae were applied to a chronically infected, parasitemic C. felis donor cat (Felis catus) and allowed to feed to repletion. Engorged larvae were collected and held through ecdysis. Three cats were each infested with 66 A. americanum or 66 D. variabilis emerged nymphs. Cytauxzoon felis infections in principal cats were determined by clinical signs and detection of circulating parasite by blood smear and PCR evaluation.

Results: Clinical signs of cytauxzoonosis were observed in cats infested with A. americanum nymphs beginning 12-15 days post-infestation (dpi). The same cats were PCR positive on 12-14 dpi; piroplasms were evident in blood smears at 16 dpi, and macrophage schizonts were observed in stained spleen impression smears in two animals at necropsy. Cats infested with acquisition-fed D. variabilis nymphs remained clinically normal and did not develop detectable parasitemia over the course of the study as determined by blood smear and PCR.

Conclusions: Cytauxzoon felis was successfully transmitted to domestic cats by A. americanum nymphs acquisition-fed as larvae on the donor cat. However, we were not able to transmit C. felis to healthy domestic cats with D. variabilis nymphs that were simultaneously acquisition-fed on the same donor cat. Results from this study suggest that larval and nymphal A. americanum likely play important roles in natural transmission cycles of C. felis.
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http://dx.doi.org/10.1186/s13071-018-3276-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6330474PMC
January 2019

Efficacy of a topical formulation of selamectin plus sarolaner against induced infestations of Amblyomma americanum on cats and prevention of Cytauxzoon felis transmission.

Vet Parasitol 2019 Jun 11;270 Suppl 1:S31-S37. Epub 2018 Nov 11.

Zoetis, Veterinary Medicine Research and Development, Kalamazoo, MI 49007, USA.

Cytauxzoonosis, caused by infection with Cytauxzoon felis, is the most severe tick-borne disease of cats. The purpose of our study was to determine the efficacy of selamectin (6.0 mg/kg) plus sarolaner (1.0 mg/kg) formulated in combination (Revolution® Plus / Stronghold Plus, Zoetis) applied topically once a month on cats for three months against induced infestations of Amblyomma americanum adults and to evaluate the effectiveness of the product in preventing the transmission of C. felis. This study was conducted in two phases. Sixteen cats were dosed with selamectin/sarolaner or a placebo (vehicle control) on Days 0, 28, and 56. In phase 1, each cat was infested with 50 (±5) unfed adult A. americanum on Day 4 and tick counts were conducted on Day 6 (48 h post infestation) and Day 7 (72 h post infestation) to evaluate acaricidal efficacy. In phase 2, to confirm acaricidal efficacy and evaluate prevention of C. felis transmission, each cat was infested on Day 60 with 50 (±5) adult A. americanum acquisition fed as nymphs on two C. felis-infected donor cats. Tick counts were conducted on Day 62 (48 h post infestation) and Day 63 (72 h post infestation). Blood samples were collected on Days -9, 60, 70, 76, and 90 and tested for infection with C. felis. Placebo cats were adequately infested on all count days, with least squares (geometric) mean live tick counts ranging from 34.0 (28.8) to 46.1 (46.0). Treatment reduced the least squares (geometric) mean counts compared to placebo by 27.1 (32.1)% and 90.4 (96.8)% on Days 6 and 7, respectively. The corresponding percent reductions were 56.4 (60.6)% and 94.7 (97.3)% on Days 62 and 63, respectively. Least squares mean counts were significantly lower in the treated group compared with the placebo group on all count days (P ≤ 0.0286). All cats were negative for C. felis by PCR prior to study start. In phase 2, seven cats in the control group and no cats in the selamectin/sarolaner group became infected with C. felis (P = 0.0017). Topical treatment with selamectin/sarolaner was >90% effective in reducing A. americanum tick counts 72 h after infestation and prevented the transmission of C. felis from infected ticks following the third of three monthly treatments. Revolution Plus / Stronghold Plus offers an option for the control of A. americanum infestations on cats and for preventing the transmission of C. felis to cats.
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http://dx.doi.org/10.1016/j.vetpar.2018.10.018DOI Listing
June 2019

Increased prevalence of Dirofilaria immitis antigen in canine samples after heat treatment.

Vet Parasitol 2014 Nov 28;206(1-2):67-70. Epub 2014 Mar 28.

Department of Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK, United States. Electronic address:

Canine serum samples may contain factors that prevent detection of antigen of Dirofilaria immitis on commercial assays, precluding accurate diagnosis. To determine the degree to which the presence of blocking antibodies or other inhibitors of antigen detection may interfere with our ability to detect circulating antigen in canine samples, archived plasma and serum samples (n=165) collected from dogs in animal shelters were tested for D. immitis antigen before and after heat treatment. Negative samples were also evaluated for their ability to block detection of D. immitis antigen in a sample from a positive dog. All 165 samples were negative prior to heating, but 11/154 (7.1%) became positive after heat treatment, a conversion that was documented and quantified on spectrophotometric plate assays, and 7/165 (4.2%) samples decreased detection of antigen when mixed with a known positive sample, suggesting some blocking ability was present. An additional 103 plasma and serum samples that tested positive prior to heating also were evaluated; the optical density of 14/101 (13.9%) increased by ≥50%, and one sample by as much as 15-fold, after heat treatment. Our results suggest that canine serum and plasma samples from dogs in the southeastern United States can contain inhibitors of D. immitis antigen detection, and that prevalence estimates of heartworm infection based on these assays would benefit from heat treatment of samples prior to testing.
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http://dx.doi.org/10.1016/j.vetpar.2014.03.021DOI Listing
November 2014

Pre-treatment with heat facilitates detection of antigen of Dirofilaria immitis in canine samples.

Vet Parasitol 2014 Jun 18;203(1-2):250-2. Epub 2014 Jan 18.

Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK, United States.

Diagnosis of Dirofilaria immitis infection in dogs is largely dependent on detection of antigen in canine serum, plasma, or whole blood, but antigen may be bound in immune complexes and thus not detected. To develop a model for antigen blocking, we mixed serum from a microfilaremic, antigen-positive dog with that of a hypergammaglobulinemic dog not currently infected with D. immitis and converted the positive sample to antigen-negative; detection of antigen was restored when the mixed sample was heat-treated, presumably due to disruption of antigen/antibody complexes. A blood sample was also evaluated from a dog that was microfilaremic and for which microfilariae were identified as D. immitis by morphologic examination. Antigen of D. immitis was not detected in this sample prior to heating but the sample was strongly positive after heat treatment of whole blood. Taken together, our results indicate that blood samples from some dogs may contain factors that inhibit detection of antigen of D. immitis, and that heat treatment of these samples prior to testing could improve the sensitivity of these assays in some patients.
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http://dx.doi.org/10.1016/j.vetpar.2014.01.007DOI Listing
June 2014

Merogonic stages of Theileria cervi in mule deer (Odocoileus hemionus).

J Vet Diagn Invest 2013 Sep;25(5):662-5

1Jason Wood, Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, 250 McElroy Hall, Stillwater, OK 74078.

In February 2012, 12 farmed mule deer (Odocoileus hemionus) were moved from a facility in southwestern Oklahoma to a facility in southeastern Oklahoma that housed 100 farmed white-tailed deer (Odocoileus virginianus). Between the third and fifth weeks, 9 of the 12 mule deer had died, 4 of which were submitted for necropsy. The deer were heavily infested with Amblyomma americanum (lone star ticks). Hematologic data from 1 deer revealed severe anemia, leukocytosis, and intraerythrocytic hemoparasites consistent with Theileria spp. Microscopically, the liver, lymph nodes, and spleen contained multifocally distributed, enlarged monocytic cells whose cytoplasm was replaced by developing meronts in various stages of merogony. It appears that, upon arrival, the Theileria cervi-naïve mule deer became infested with large numbers of Theileria-infected lone star ticks leading to massive exposure of the mule deer to sporozoites of the protozoan, resulting in an acute hemolytic crisis and fatalities. The merogonic stages of T. cervi are also described. The lack of earlier reports of merogony may be due to the fact that only a single, short-lived, merogonic cycle follows exposure to sporozoites and thus merogonic stages are demonstrable for only a short period. Polymerase chain reaction testing of paraffin-embedded tissue yielded a 507-bp amplicon sequence that was 100% identical with the sequence of T. cervi previously reported from white-tailed deer in Oklahoma and from elk in Wisconsin and Indiana.
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http://dx.doi.org/10.1177/1040638713501173DOI Listing
September 2013

Genetic diversity of Hepatozoon spp. in coyotes from the south-central United States.

J Parasitol 2013 Apr 27;99(2):375-8. Epub 2012 Aug 27.

Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Stillwater, Oklahoma 74078, USA.

To better define the strains and species of Hepatozoon that infect coyotes in the south-central United States, whole blood and muscle samples were collected from 44 coyotes from 6 locations in Oklahoma and Texas. Samples were evaluated by a nested polymerase chain reaction (PCR) using primers amplifying a variable region of the apicomplexan 18S rRNA gene as well as histopathology (muscle only) for presence of tissue cysts. Hepatozoon spp. infections were identified in 79.5% (35/44) of coyotes tested including 27 of 44 (61.4%) whole blood samples and 17 of 44 (38.6%) muscle samples tested by PCR and 23 of 44 (52.3%) muscle samples evaluated by histological examination. Analysis revealed 19 distinct sequences comprising 3 major clusters of Hepatozoon spp., i.e., 1 most closely related to Hepatozoon americanum, another most closely related to Hepatozoon canis , and the third an intermediate between the 2 groups. The diversity of Hepatozoon spp. in wild canids appears greater than previously recognized and warrants further investigation.
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http://dx.doi.org/10.1645/GE-3104.1DOI Listing
April 2013

Hepatozoon spp infections in the United States.

Vet Clin North Am Small Anim Pract 2011 Nov;41(6):1221-38

Department of Veterinary Pathobiology, Oklahoma State University Center for Veterinary Health Sciences, 250 McElroy Hall, Stillwater, OK 74078, USA.

Two Hepatozoon spp are recognized as parasites of domestic dogs in the United States, H. canis and H. americanum. H. canis was first described in India in 1905 and has been documented in many areas of the world, although not definitively identified in North America until recently. H. americanum, causing American canine hepatozoonosis, was first documented in a coyote in 1978 and is now considered an emerging etiologic agent of disease in domestic dogs throughout the United States. The authors review current knowledge of canine hepatozoonosis caused by H. canis and H. americanum and elaborate on more recent research findings.
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http://dx.doi.org/10.1016/j.cvsm.2011.08.006DOI Listing
November 2011

Novel Hepatozoon in vertebrates from the southern United States.

J Parasitol 2011 Aug 25;97(4):648-53. Epub 2011 Feb 25.

Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, Oklahoma 74074, USA.

Novel Hepatozoon spp. sequences collected from previously unrecognized vertebrate hosts in North America were compared with documented Hepatozoon 18S rRNA sequences in an effort to examine phylogenetic relationships between the different Hepatozoon organisms found cycling in nature. An approximately 500-base pair fragment of 18S rDNA common to Hepatozoon spp. and some other apicomplexans was amplified and sequenced from the tissues or blood of 16 vertebrate host species from the southern United States, including 1 opossum (Didelphis virginiana), 2 bobcats (Lynx rufus), 1 domestic cat (Felis catus), 3 coyotes (Canis latrans), 1 gray fox (Urocyon cinereoargenteus), 4 raccoons (Procyon lotor), 1 pet boa constrictor (Boa constrictor imperator), 1 swamp rabbit (Sylvilagus aquaticus), 1 cottontail rabbit (Sylvilagus floridanus), 4 woodrats (Neotoma fuscipes and Neotoma micropus), 3 white-footed mice (Peromyscus leucopus), 8 cotton rats (Sigmodon hispidus), 1 cotton mouse (Peromyscus gossypinus), 1 eastern grey squirrel (Sciurus carolinensis), and 1 woodchuck (Marmota monax). Phylogenetic analyses and comparison with sequences in the existing database revealed distinct groups of Hepatozoon spp., with clusters formed by sequences obtained from scavengers and carnivores (opossum, raccoons, canids, and felids) and those obtained from rodents. Surprisingly, Hepatozoon spp. sequences from wild rabbits were most closely related to sequences obtained from carnivores (97.2% identical), and the sequence from the boa constrictor was most closely related to the rodent cluster (97.4% identical). These data are consistent with recent work identifying prey-predator transmission cycles in Hepatozoon spp. and suggest this pattern may be more common than previously recognized.
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http://dx.doi.org/10.1645/GE-2672.1DOI Listing
August 2011

Treatment of Hepatozoon americanum infection: review of the literature and experimental evaluation of efficacy.

Vet Ther 2010 ;11(4):E1-8

Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK, USA.

There is no labeled treatment for dogs with American canine hepatozoonosis (ACH), but the drug therapies discussed in this article, although not rapidly curative, may be successful in alleviating acute clinical signs, prolonging life, reducing the number of clinical relapses, and enhancing quality of life. This article also describes a pilot trial conducted to assess the efficacy of a novel treatment approach with ponazuril as a stand-alone parasiticide administered for 4 weeks without follow-up decoquinate treatment. Although extended ponazuril treatment in combination with NSAID administration did ameliorate acute clinical signs associated with ACH, the parasite was not completely cleared with this treatment protocol alone. Long-term decoquinate therapy remains a critical component of successful treatment of ACH.
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October 2013

The increasing recognition of rickettsial pathogens in dogs and people.

Trends Parasitol 2010 Apr 6;26(4):205-12. Epub 2010 Mar 6.

Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.

Dogs and people are exposed to and susceptible to infection by many of the same tick-borne bacterial pathogens in the order Rickettsiales, including Anaplasma phagocytophilum, Ehrlichia canis, E. chaffeensis, E. ewingii, Rickettsia rickettsii, R. conorii, and other spotted fever group rickettsiae. Recent findings include descriptions of novel Ehrlichia and Rickettsia species, recognition of the occurrence and clinical significance of co-infection, and increasing awareness of Rhipicephalus sanguineus-associated diseases. Newer molecular assays are available, although renewed efforts to encourage their use are needed. This review highlights the ecology and epidemiology of these diseases, and proposes avenues for future investigation.
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http://dx.doi.org/10.1016/j.pt.2010.01.007DOI Listing
April 2010

Detection of Babesia gibsoni and the canine small Babesia 'Spanish isolate' in blood samples obtained from dogs confiscated from dogfighting operations.

J Am Vet Med Assoc 2009 Sep;235(5):535-9

Department of Veterinary Pathobiology, Center for Veterinary Health Science, Oklahoma State University, Stillwater, OK 74078, USA.

Objective: To determine the prevalence of Babesia gibsoni infection in dogs that were confiscated from dogfighting operations.

Design: Cross-sectional study.

Animals: 157 pit bull-type dogs that were confiscated as part of dogfighting prosecution cases in Iowa, Michigan, Mississippi, Ohio, Pennsylvania, Virginia, and Washington and 218 randomly selected animal shelter dogs with no known history of dogfighting.

Procedures: Blood samples collected from confiscated dogs were tested for infection with B gibsoni by use of a nested PCR assay. Samples that yielded positive results underwent DNA sequencing to confirm infection with B gibsoni. Control blood samples collected from 218 randomly selected dogs in animal shelters (ie, dogs that had no known involvement in dogfighting events) were also analyzed.

Results: Results of nested PCR assays indicated that 53 of 157 (33.8%) confiscated dogs were infected with B gibsoni; 1 (0.6%) dog was infected with the canine small Babesia 'Spanish isolate' (also known as Theileria annae). To the authors' knowledge, this is the first report of infection with this small Babesia 'Spanish isolate' in a North American dog. Dogs with scars (indicative of fighting) on the face, head, and forelimbs were 5.5 times as likely to be infected with B gibsoni as were dogs without scars. Of the control dogs, 1 (0.5%) pit bull-type dog was infected with B gibsoni.

Conclusions And Clinical Relevance: Results indicated that B gibsoni is a common parasite of dogs confiscated from dogfighting operations and suggested that dogs with a history of fighting should be evaluated for infection with B gibsoni.
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http://dx.doi.org/10.2460/javma.235.5.535DOI Listing
September 2009

Experimental transmission of Hepatozoon americanum to New Zealand White rabbits (Oryctolagus cuniculus) and infectivity of cystozoites for a dog.

Vet Parasitol 2009 Oct 6;164(2-4):162-6. Epub 2009 Jun 6.

Oklahoma State University, Center for Veterinary Health Sciences, Department of Veterinary Pathobiology, Stillwater, OK 74078, USA.

Inflammatory lesions containing parasitic cystozoites developed in multiple organs and tissues of laboratory-raised Oryctolagus cuniculus that were administered approximately 100 sporulated oocysts of Hepatozoon americanum (Oklahoma isolate, GenBank accession AF176836) orally. The predominantly granulomatous inflammatory lesions were detected histologically 8 weeks after exposure to oocysts. Cystozoites, recognized by cresent-shaped, uninucleated bodies surrounded by an accumulation of globular, PAS-positive polysaccharide material, were evident within macrophages as monozoic and dizoic cysts. Neither meronts nor gamonts were detected in any of the laboratory-raised lagomorphs during the 24-week observation period. Nested PCR assay of rabbit tissues for a 488 bp fragment of the 18S rRNA Hepatozoon spp. gene was positive at 8 and 24 weeks post-exposure. The sequence was 97.1% similar with sequence from the H. americanum carrier used to infect ticks. A Hepatozoon-free dog fed tissues from the 24-week post-exposure rabbit principal developed American canine hepatozoonosis. Gamonts were first detected 5 weeks after the dog ingested the rabbit tissues containing cystozoites. PCR assay of blood from the dog was positive for the Hepatozoon spp. gene fragment. Sequencing confirmed that the parasite in the dog was H. americanum.
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http://dx.doi.org/10.1016/j.vetpar.2009.05.028DOI Listing
October 2009

New developments in canine hepatozoonosis in North America: a review.

Parasit Vectors 2009 Mar 26;2 Suppl 1:S5. Epub 2009 Mar 26.

Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK, USA.

Canine hepatozoonosis is caused by Hepatozoon canis and Hepatozoon americanum, apicomplexan parasites transmitted to dogs by ingestion of infectious stages. Although the two agents are phylogenetically related, specific aspects, including characteristics of clinical disease and the natural history of the parasites themselves, differ between the two species. Until recently, H. canis infections had not been clearly documented in North America, and autochthonous infection with H. americanum has yet to be reported outside of the southern United States. However, recent reports demonstrate H. canis is present in areas of North America where its vector tick, Rhipicephalus sanguineus, has long been endemic, and that the range of H. americanum is likely expanding along with that of its vector tick, Amblyomma maculatum; co-infections with the two organisms have also been identified. Significant intraspecific variation has been reported in the 18S rRNA gene sequence of both Hepatozoon spp.-infecting dogs, suggesting that each species may represent a complex of related genogroups rather than well-defined species. Transmission of H. americanum to dogs via ingestion of cystozoites in muscle of infected vertebrates was recently demonstrated, supporting the concept of predation as a means of natural transmission. Although several exciting advances have occurred in recent years, much remains to be learned about patterns of infection and the nature of clinical disease caused by the agents of canine hepatozoonosis in North America.
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http://dx.doi.org/10.1186/1756-3305-2-S1-S5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2679397PMC
March 2009

Diagnosis of canine Hepatozoon spp. infection by quantitative PCR.

Vet Parasitol 2008 Oct 17;157(1-2):50-8. Epub 2008 Jul 17.

Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849, USA.

Hepatozoon (H.) americanum and H. canis are the etiological agents of canine hepatozoonosis, a disease that is found worldwide and is also prevalent in the southeastern United States. Current laboratory diagnosis of canine hepatozoonosis caused by H. americanum is usually dependent on visual identification of Hepatozoon "onion skin cysts" in muscle biopsies, an approach that requires invasive sampling and can result in false negatives. We have developed a diagnostic method for detection of Hepatozoon spp. DNA that integrates nucleic acid extraction with extensive agitation to maximize DNA extraction efficiency. The DNA extracted from canine EDTA-whole blood is subjected to real-time PCR, and fluorescence resonance energy transfer (FRET) probes detect a signature polymorphism in the amplified DNA. This PCR method amplifies a fragment of the Hepatozoon 18S rDNA gene, detects as few as 7 genomic copies of Hepatozoon spp. per ml of blood with high specificity, and differentiates between H. americanum and H. canis amplicons. A surprising 300-fold increase of H. americanum 18S rDNA targets occurred during 3-0 days of storage of positive blood specimens. Examination of 614 EDTA-blood samples submitted mostly from the southeastern Unites States from dogs with suspected hepatozoonosis identified H. americanum in 167 samples (27.2%). An additional 14 samples (2.3%) were positive for H. canis, and 14 samples (2.3%) were positive for both H. americanum and H. canis. These results suggest that the Hepatozoon spp. 18S rDNA quantitative PCR may be a valuable tool that can improve diagnosis and therapy of canine hepatozoonosis.
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http://dx.doi.org/10.1016/j.vetpar.2008.06.027DOI Listing
October 2008

Diversity of Hepatozoon species in naturally infected dogs in the southern United States.

Vet Parasitol 2008 Jul 7;154(3-4):220-5. Epub 2008 Apr 7.

Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USA.

Hepatozoon americanum is a protozoan that causes American canine hepatozoonosis (ACH) in the southern United States; Hepatozoon canis, the causative agent of canine hepatozoonosis in Africa, Asia, Europe, and South America, has not previously been definitively identified in dogs in the United States. To characterize the diversity of Hepatozoon spp. in domestic dogs from Oklahoma, blood samples collected from dogs residing in an endemic area of the state, clinical cases presented to veterinarians with symptoms of ACH, and dogs housed at a local shelter were evaluated by a nested PCR designed to amplify a variable region of the 18S rRNA gene of blood ampicomplexa, including Hepatozoon spp. Hepatozoon sequences recovered from a dog from an area where ACH is endemic, from clinically ill dogs, and from one shelter dog most closely resembled H. americanum. However, two other shelter dogs had evidence of infection with H. canis or a closely related organism. A subsequent review of real-time PCR results from the Molecular Diagnostics Laboratory at Auburn University revealed that the majority of samples submitted from dogs from across the United States which tested positive for Hepatozoon spp. had H. americanum. However, some submissions were also found which contained DNA sequence of H. canis. Mixed H. americanum and H. canis-like infections also were detected. Our data suggest that H. americanum, H. canis, as well as H. canis-like organisms are present and may cause disease in dogs in the southern U.S.
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http://dx.doi.org/10.1016/j.vetpar.2008.03.027DOI Listing
July 2008

Infectivity of Hepatozoon americanum cystozoites for a dog.

Vet Parasitol 2008 Jun 29;154(1-2):148-50. Epub 2008 Feb 29.

Oklahoma State University (OSU), College of Veterinary Health Sciences (CVHS), Department of Veterinary Pathobiology, Room 250 McElroy Hall, Stillwater, OK 74078, United States.

Hepatozoon americanum cystozoites from experimentally infected, laboratory-raised rodents were fed to a Hepatozoon-free dog. Gamonts were detected by examination of blood smear 42 and 56 days post-exposure. PCR analysis of blood was positive for the 18S rRNA Hepatozoon gene on days gamonts were demonstrated. Meronts were detected histologically in a skeletal muscle biopsy 90 days after ingestion of cystozoites. Sequencing confirmed that the parasite in the dog was H. americanum. Xenodiagnosis was conducted by replete feeding of Ambylomma maculatum larvae on the dog; 40 days after detachment, sporulated oocysts were recovered from recently molted nymphs.
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http://dx.doi.org/10.1016/j.vetpar.2008.02.026DOI Listing
June 2008

Experimental transmission of Hepatozoon americanum to rodents.

Vet Parasitol 2008 Feb 4;151(2-4):164-9. Epub 2007 Nov 4.

Oklahoma State University, College of Veterinary Health Sciences, Department of Veterinary Pathobiology, Room 250 McElroy Hall, Stillwater, OK 74078-2007, United States.

Laboratory-raised cotton rats (Sigmodon hispidus), outbred white mice (Mus musculus), and C57BL/6J-Lystbg-J/J mice (M. musculus) that were administered approximately 50 sporulated oocysts of Hepatozoon americanum (AF176836) by gavage developed inflammatory lesions containing parasitic cystozoites in cardiac and skeletal muscle, kidney, and lung. Sprague-Dawley rats (Rattus norvegicus) similarly exposed showed no evidence of infection. Cystozoites were first detected by histopathologic examination four weeks after exposure to oocysts. Globular, PAS-positive material accumulated around the cystozoites as the duration of infection lengthened. Nested PCR analysis of tissues collected 16 weeks post-exposure was positive for the 18S rRNA Hepatozoon sp. gene and the DNA sequence of the fragment amplified was 99.6% and 99.8% identical to H. americanum sequences previously reported from naturally-infected dogs (AF176836 and AY864676, respectively). Merogonous and gamontogonous stages of the parasite were not detected in any of the cystozoite-infected rodents.
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http://dx.doi.org/10.1016/j.vetpar.2007.10.017DOI Listing
February 2008

Field survey of rodents for Hepatozoon infections in an endemic focus of American canine hepatozoonosis.

Vet Parasitol 2007 Nov 22;150(1-2):27-32. Epub 2007 Oct 22.

Oklahoma State University, Center for Veterinary Health Sciences, Department of Veterinary Pathobiology, Room 250, McElroy Hall, Stillwater, OK 74078-2007, United States.

Eighteen of 31 (58%) cotton rats (Sigmodon hispidus) and 8 of 24 (33.3%) white-footed mice (Peromyscus leucopus) that were wild-trapped from 4 American canine hepatozoonosis endemic sites in Oklahoma were infected with Hepatozoon species. The predilection organ for merogony of the Hepatozoon species in cotton rats was the liver. Meronts were not detected in any of the white-footed mice. A 488 bp DNA fragment that includes a variable region of the 18S rRNA Hepatozoon gene amplified from blood or tissue of these infected animals. Sequences from eight cotton rats were 100% identical to each other as were sequences from three white-footed mice 100% identical to each other. The cotton rat sequence and the white-footed mouse sequence were 98.8% identical, differing in 6 bp of the 488 bp fragment. The DNA sequence from cotton rats was 97.7% identical to a Hepatozoon sp. described in a large bandicoot rat from Thailand and 97.5% identical to a Hepatozoon sp. in a bank vole from Brazil. The sequence from white-footed mice was 98.6% identical to the bandicoot rat sequence and 98.4% identical to the bank vole sequence. However, the sequences were only 90.6% (cotton rat) and 91.4% (white-footed mouse) identical to H. americanum. These findings suggest that the rodents are obligate intermediate hosts for distinct Hepatozoon spp., but not H. americanum.
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http://dx.doi.org/10.1016/j.vetpar.2007.08.050DOI Listing
November 2007
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