Molecular Detection, Phylogenetic Analysis, and Genetic Diversity of Theileria annulata, Babesia bigemina, and Anaplasma marginale in Cattle in Three Districts of Egypt.

BACKGROUND: Under the poor hygienic conditions, tick-borne pathogens cause severe economic losses to the cattle industry. PURPOSE: The current study investigated the presence of Theileria annulata, Babesia bigemina, and Anaplasma marginale, the most relevant tick-borne pathogens in cattle, in 3 provinces of Egypt utilizing species-specific PCR assays. METHODS: PCR was conducted, on bovine blood specimens, using primers targeting the T. annulata merozoite-piroplasm surface antigen (Tams1, 768 bp), A. marginale major surface protein-1b gene (msp1b, 265 bp), and B. bigemina small subunit ribosomal RNA gene (SSrRNA, 543 bp). RESULTS: PCR findings revealed overall prevalences of T. annulata, B. bigemina, and A. marginale as 22.0% (33/150), 19.33% (29/150), and 10.6% (16/150), respectively. The co-infection with two or three pathogens was detected in 20.0% (30/150) of examined specimens. Sequence analyses indicated that T. annulata and A. marginale varied from those of corresponding GenBank sequences revealing percent identities ranging from 90.68 to 97.75% and from 94.98 to 98.63%, respectively. On the other hand, the obtained B. bigemina sequences showed a high similarity with those previously reported in GenBank with a percent identity ranging from 98.85 to 100%. CONCLUSION: T. annulata was the most prevalent tick-borne pathogen in examined bovine specimens. The genetic diversity of markers used for identification of T. annulata and A. marginale should be highly considered.

In vitro cultivation of Babesia duncani (Apicomplexa: Babesiidae), a zoonotic hemoprotozoan, using infected blood from Syrian hamsters (Mesocricetus auratus).

Human babesiosis, a tick-borne disease similar to malaria, is most often caused by the hemoprotozoans Babesia divergens in Europe, and Babesia microti and Babesia duncani in North America. Babesia microti is the best documented and causes more cases of human babesiosis annually than all other agents combined. Although the agents that cause human babesiosis are considered high-risk pathogens in transfusion medicine, federally licensed diagnostics are lacking for B. duncani in both the USA and Canada. Thus, there has been a need to develop and validate diagnostics specifically for this pathogen. In this study, B. duncani (WA1 isolate) was cultivated in vitro from Syrian hamster (Mesocricetus auratus) infected blood. We hypothesized HL-1 media with supplements would result in B. duncani propagating at higher levels in culture than supplemented M199 similar to the medium the parasite was originally cultivated with in 1994. We were unable to recreate Thomford's cultivation results with the M199 medium but supplemented HL-1 medium was able to successfully establish continuous culture. We further hypothesized that RBC from species other than hamsters would support B. duncani in vitro. However, rat, mouse, horse, and cow RBC did not support continuous culture of the parasite. Culture stocks of B. duncani were deposited at BEI Resources and are now commercially available to the scientific community to further research. The cultured parasite developed in this study was instrumental in the adaptation of B. duncani continuous culture to human RBC.

Effect of high concentrations of lufenuron, pyriproxfen and hydroprene on Rhipicephalus (Boophilus) annulatus.

The activity of high doses of three insect growth regulators (IGRs), lufenuron (MATCH®), pyriproxfen® and hydroprene (Gentrol®), were tested on Rhipicephalus(Boophilus) annulatus adult females, eggs and larvae. Different concentrations of the IGRs were tested on eggs, larvae and adult ticks through immersion, larval packet and adult immersion bioassays, respectively. The tested IGRs did not show adulticidal activity against female ticks even at very high concentration. However, both hydroprene and pyriproxfen caused a significant decrease (P<0.05) in the reproductive indices of adult female ticks. Both lufenuron and pyriproxefen showed considerable ovicidal activity delaying the hatchability of the treated eggs until the 21st day and decreasing the hatchability percentages to 37.7% and 60.6% at concentrations ≥10X and ≥4X, respectively. Lufenuron (≥10X dose), hydroprene (≥4X dose) and pyriproxyfen (≥4X dose) induced highly significant larvicidal activity as they caused 100% mortality after 72 h of exposure. The oxidative profile of the hydroprene treated ticks had decreased glutathione peroxidase and increased malonaldehyde in comparison to the other IGR- treated and control untreated ticks. It is concluded that the IGRs did not show R. annulatus adulticidal effect, however, the deposited egg mass and its hatching percent decreased significantly when treated with hydroprene and pyriproxfen. The tested IGRs showed larvicidal activity against R. (B.) annulatus.

The Babesia divergens Asia Lineage Is Maintained through Enzootic Cycles between Ixodes persulcatus and Sika Deer in Hokkaido, Japan.

Parasites of the Babesiadivergens Asia lineage, which are closely related to B. divergens in Europe and Babesia sp. strain MO1 in the United States, were recently reported in sika deer (Cervus nippon) in eastern Japan. To identify the tick vector(s) for this parasite, we conducted a field survey in Hokkaido, Japan, where the infection rate in sika deer is the highest in the country. A specific PCR system which detects and discriminates between lineages within B. divergens and between those lineages and Babesia venatorum showed that Ixodes persulcatus (11/822), but not sympatric Ixodes ovatus (0/595) or Haemaphysalis sp. (0/163) ticks, carried B. divergens Asia lineage. Genomic DNA was archived from salivary glands of partially engorged I. persulcatus females and three isolates of B. divergens Asia lineage were newly described. The 18S rRNA gene sequence of the isolates formed the Asia lineage cluster with those previously described in sika deer isolates. One salivary gland also contained parasites of Babesia microti U.S. lineage, which were subsequently isolated in a hamster in vivoB. venatorum (strain Etb5) was also detected in one I. persulcatus tick. The 18S rRNA sequence of Etb5 was 99.7% identical to that of B. venatorum (AY046575) and was phylogenetically positioned in a taxon composed of B. venatorum isolates from Europe, China, and Russia. The geographical distribution of I. persulcatus is consistent with that of B. divergens in sika deer in Japan. These results suggest that I. persulcatus is a principal vector for B. divergens in Japan and Eurasia, where I. persulcatus is predominantly distributed.IMPORTANCE The Babesiadivergens Asia lineage of parasites closely related to B. divergens in Europe and Babesia sp. MO1 in the United States was recently reported in Cervus nippon in eastern Japan. In this study, specific PCR for the Asia lineage identified 11 positives in 822 host-seeking Ixodes persulcatus ticks, a principal vector for many tick-borne disease agents. Gene sequences of three isolates obtained from DNA in salivary glands of female ticks were identical to each other and to those in C. nippon We also demonstrate the coinfection of B. divergens Asia lineage with Babesia microti U.S. lineage in a tick salivary gland and, furthermore, isolated the latter in a hamster. These results suggest that I. persulcatus is the principal vector for B. divergens as well as for B. microti, and both parasites may be occasionally cotransmitted by I. persulcatus This report will be important for public health, since infection may occur through transfusion.

Diversity of Borrelia spirochetes and other zoonotic agents in ticks from Kyiv, Ukraine.

Lyme borreliosis (LB) is caused by tick-borne spirochetes of the Borrelia burgdorferi sensu lato complex. LB is the most prevalent vector-borne illness in Ukraine, but current data on the prevalence of LB pathogens in their tick vector, Ixodes ricinus, are lacking. I. ricinus ticks may also carry Borrelia miyamotoi, an emerging relapsing fever group spirochete that has been implicated in human illness. Despite its zoonotic potential, the prevalence of B. miyamotoi in ticks has not been examined in Ukraine. Similarly, data on the prevalence of other important tick-borne pathogens, Anaplasma phagocytophilum, Babesia spp., Bartonella spp., Francisella tularensis, and Rickettsia spp., in ixodid ticks are scarce or even absent. Thus, the overall objective of this study was to investigate the prevalence of these tick-borne pathogens in questing I. ricinus and Dermacentor reticulatus ticks collected in recreational parks of Kyiv, the most densely populated city of Ukraine. A total of 182 adult I. ricinus, 98 nymphal I. ricinus, and 98 adult D. reticulatus ticks were molecularly analyzed for the presence of these pathogens. As a result, the study shows a greater diversity of Borrelia genospecies in questing I. ricinus ticks than previously reported. The most prevalent genospecies in adult I. ricinus ticks were B. afzelii (7.7%), followed by B. burgdorferi sensu stricto (s.s.) (2.2%) and B. garinii (0.5%). In contrast, B. burgdorferi s.s. was most dominant in unfed I. ricinus nymphs (67.3%). Moreover, B. afzelii was detected in 11.2% of nymphs, but only 1.0% of nymphal ticks were positive for B. garinii and B. valaisiana. Importantly, this study provides the first record of B. miyamotoi detected in I. ricinus ticks from Ukraine (1.1%). Furthermore, the report is also the first to document other vector-borne pathogens, Bartonella henselae, Rickettsia conorii, and Rickettsia mendelii, in ixodid ticks from Ukraine. In summary, this work offers the latest data on the diversity and prevalence of the important zoonotic tick-borne agents in questing ticks from Kyiv, Ukraine. The data will help to better gauge the risk associated with vector-borne infections to which residents and guests of Ukraine's capital may be exposed.

Genetic variability of cloned Cytauxzoon felis ribosomal RNA ITS1 and ITS2 genomic regions from domestic cats with varied clinical outcomes from five states.

Cytauxzoon felis is a tick-borne hemoparasite that causes cytauxzoonosis in domestic cats in the United States. Historically, feline cytauxzoonosis was reported to be nearly always fatal. However, increasing evidence of cats surviving acute infection and/or harboring a chronic, subclinical infection has suggested the existence of different C. felis strains that may vary in pathogenicity. In this study, the intraspecific variation of the C. felis first and second ribosomal RNA internal transcribed spacer (ITS1, ITS2) regions was assessed for any clinical outcome or geographic associations. Sequence data were obtained for 122C. felis ITS1 and ITS2 clones from 41 domestic cat blood samples from Arkansas, Kansas, Missouri, Oklahoma, and Texas. Seven previously reported ITS1 region sequences were found, and a previously undescribed 23-bp insert was detected in cloned ITS1 sequences from a domestic cat in Missouri and two cats in Oklahoma. Four previously reported ITS2 region sequences were identified, and a 40-bp insert similar to that previously reported in C. felis of a domestic cat from Arkansas and pumas was detected in 18 cloned C. felis sequences from 12 domestic cats. One clone contained both the 23-bp insert and 40-bp insert within the ITS1 and ITS2 regions, respectively. Combined ITS1 and ITS2 sequence genotypes revealed that C. felis sequences from 27 cats (72/122 clones) corresponded to four previously described genotypes, ITSa, ITSc, ITSd, and ITSn. Five clones with the novel 23-bp insert from three cat isolates represented two new genotypes, ITSaa and ITSbb. Genotypes ITScc, ITSdd, ITSee, ITSff, ITSgg, and ITShh denoted 13 clones that matched prior sequences but had no previously assigned genotype. Genotypes ITSii through ITStt comprised 32 clones that were similar to, but did not exactly match, previously described genotypes. Twenty-five cats had C. felis infections with multiple ITS genotypes. Considerable C. felis genetic diversity was revealed with no significant geographic or clinical outcome associations.

Recovery of bovine Babesia spp. after long-term cryostorage and comparison of bovine donor erythrocytes and serum.

Cultured Babesia bovis and Babesia bigemina were recovered from liquid nitrogen storage nearly 30 years after they were cryopreserved. Four cattle were compared as donors of erythrocytes and serum for microaerophilous stationary phase (MASP) cultures for recovery of B. bigemina. Erythrocytes and serum from only one (#913) of the four animals supported growth of B. bigemina. Two B. bigemina (frozen in 1986 and 1987) and two B. bovis (both frozen in 1986) cryostocks were recovered from liquid nitrogen storage and all four recovered and thrived in #913 erythrocytes and serum. In the third passage after recovery, B. bovis cultures were cryopreserved. Six months later they were successfully recovered using #913 erythrocytes and serum. This study shows that B. bovis and B. bigemina stored nearly 30 years in liquid nitrogen can be successfully recovered in the MASP system. This study also confirms previous observations that selection of a suitable bovine donor of erythrocytes and serum is critical to the success of the culture.

Molecular and morphological characterization of a haemogregarine in the alligator snapping turtle, Macrochelys temminckii (Testudines: Chelydridae).

A severely underweight alligator snapping turtle Macrochelys temminckii Troost in Harlan, 1835, was found near Tyler, Texas, and taken to the Caldwell Zoo. Blood films were submitted to Texas A&M University, College Station, Texas, for morphological and molecular identification of haemogregarine-like inclusions in the red blood cells. Intraerythrocytic Haemogregarina sp. forms were found on microscopic examination at a parasitemia of <1 %. The morphology and morphometric data for the forms indicate similarity to Haemogregarina macrochelysi n. sp. Telford et al., 2009, previously reported in alligator snapping turtles in Florida and Georgia, but two characteristic stage forms were not shared between H. macrochelysi n. sp. and the parasite found in this report. The haemogregarine 18S ribosomal RNA gene (1555-bp fragment) was amplified and cloned, and five clones sequenced. The sequences were deposited in the NCBI GenBank database. All five showed ∼96 % identity to Haemogregarina balli Paterson and Desser, 1976, Hepatozoon sp., and Hemolivia stellata Petit et al., 1990. A 774-bp segment shared 98-99 % identity with the corresponding Haemogregarina sp. rDNA sequence (KR006985) from Caspian turtles (Mauremys caspica McDowell, 1964) in Iran. A neighbor-joining phylogenetic tree generated from aligned sequences from the clones, 26 hematozoa, Adelina dimidiata Schneider, 1875, and Cryptosporidium serpentis Levine, 1980, revealed the cloned sequences clustered on their own branch within the Haemogregarina spp. clade. No genetic data are available for H. macrochelysi n. sp. at this time, so it remains unclear if this parasite in a Texas alligator snapping turtle is conspecific with H. macrochelysi n. sp.

Genotypic and phenotypic evaluation for benzimidazole resistance or susceptibility in Haemonchus contortus isolates.

Haemonchus contortus isolates were evaluated for benzimidazole (BZ) resistance or susceptibility by allele-specific PCR based on ß-tubulin isotype 1 gene polymorphisms at the F167Y, E198A, and F200Y sites. Two isolates, one presumed susceptible from wild pronghorn antelope (PH) and one known to be resistant from goats (VM), were also assayed phenotypically for BZ resistance or susceptibility in the larval development assay (Drenchrite®). The BZ EC50 was 0.198 µM (intermediate between susceptible and weak resistant) for PH with critical well 5 (intermediate between susceptible and weak resistant) and 1.456 µM (intermediate weak resistant and resistant) for VM with critical well 8.5 (resistant). Genotypically, DNA extracted from pooled VM L3 larvae in the Drenchrite® wells with the highest BZ concentration was homozygous susceptible (SS) at the F167Y and E198A sites and homozygous resistant (RR) at the F200Y site by PCR, and sequence analysis bore this out. PH L3 larvae DNA from a control well (no BZ) was SS at all three sites by PCR, confirmed by sequence analysis. All single adult worm samples (N = 21) from PH, VM, Egypt goat (EG), and a Texas llama were SS at F167Y and E198A by PCR; however, only 3 PH worms and 1 EG worm were SS at F200Y. Three additional PH worms were RS and upon cloning two clones were identified as resistant by sequencing and two as susceptible. Clones from single adult worms VM, llama, and EG samples that were RR by PCR at F200Y were sequence verified as resistant. In this study, F200Y was the most frequently found genotypic marker for BZ resistance or susceptibility in the different Haemonchus isolates.

Ixodes persulcatus Ticks as Vectors for the Babesia microti U.S. Lineage in Japan.

The U.S. lineage, one of the major clades in the Babesia microti group, is known as a causal agent of human babesiosis mostly in the northeastern and upper midwestern United States. This lineage, however, also is distributed throughout the temperate zone of Eurasia with several reported human cases, although convincing evidence of the identity of the specific vector(s) in this area is lacking. Here, the goal was to demonstrate the presence of infectious parasites directly in salivary glands of Ixodes persulcatus, from which U.S. lineage genetic sequences have been detected in Asia, and to molecularly characterize the isolates. Five PCR-positive specimens were individually inoculated into hamsters, resulting in infections in four; consequently, four strains were newly established. Molecular characterization, including 18S rRNA, ß-tubulin, and CCT7 gene sequences, as well as Western blot analysis and indirect fluorescent antibody assay, revealed that all four strains were identical to each other and to the U.S. lineage strains isolated from rodents captured in Japan. The 18S rRNA gene sequence from the isolates was identical to those from I. persulcatus in Russia and China, but the genetic and antigenic profiles of the Japanese parasites differ from those in the United States and Europe. Together with previous epidemiological and transmission studies, we conclude that I. persulcatus is likely the principal vector for the B. microti U.S. lineage in Japan and presumably in northeastern Eurasia. IMPORTANCE: The major cause of human babesiosis, the tick-borne blood parasite Babesia microti, U.S. lineage, is widely distributed in the temperate Northern Hemisphere. However, the specific tick vector(s) remains unidentified in Eurasia, where there are people with antibodies to the B. microti U.S. lineage and cases of human babesiosis. In this study, the first isolation of B. microti U.S. lineage from Ixodes persulcatus ticks, a principal vector for many tick-borne diseases, is described in Japan. Limited antigenic cross-reaction was found between the Japan and United States isolates. Thus, current serological tests based on U.S. isolates may underestimate B. microti occurrence outside the United States. This study and previous studies indicate that I. persulcatus is part of the B. microti U.S. lineage life cycle in Japan and, presumably, northeastern Eurasia. This report will be important for public health, especially since infection may occur through transfusion, and also to researchers in the field of parasitology.

Expression of bovine genes associated with local and systemic immune response to infestation with the Lone Star tick, Amblyomma americanum.

The Lone Star tick, Amblyomma americanum Linnaeus 1758 (Acari; Ixodidae), causes considerable production losses to the southern U.S. cattle industry due to reduced weight, infertility, secondary infections at bite wound sites, damaged hides, and potentially death, as these ticks tend to infest livestock in large numbers. Increasing environmental concerns, along with the potential for chemical residue in food products, have led to more emphasis on alternative tick control strategies, such as selective breeding practices and anti-tick vaccines. To enable progress toward these goals, a better understanding of bovine host immune mechanisms elicited by ticks is needed. In this study, 7 calves were phenotyped as susceptible, moderately resistant, or highly resistant to adult A. americanum ticks. Tick bite-site biopsies and blood leukocytes were collected at multiple time points throughout 3 successive tick infestations. Gene expression at tick bite-site biopsies was assessed by microarray analysis over 3 time points for each phenotype group. Quantitative reverse transcriptase-PCR expression analysis evaluated 11 candidate genes in tick bite-site biopsies, and 6 in blood leukocytes. Regression curve estimates calculated from the expression values generated by qRT-PCR in tick bite-sites identified correlations between several candidate genes. Increased expression of IGHG1, IL6, IL1α, and IL1RN in bovine tick bite-site biopsies suggests that Th2 differentiation may be important for the local bovine response to A. americanum ticks. Strong correlations in expression for IL1α and IL1ß, for IL1α and IL1RN, and for IL1α and TLR4 were found in biopsies from the tick-resistant phenotypes. The up-regulation of IL12 and IL23 in blood leukocytes from Lone Star tick-infested calves of all phenotypes suggests a possible systemic recruitment of memory T cells. This study provides novel insight concerning the bovine immune response to Lone Star ticks and a basis for future investigations to characterize the importance of these factors for tick-resistance in cattle.

Persistent babesiosis in a Rhesus macaque (Macaca mulatta) infected with a simian-human immunodeficiency virus.

BACKGROUND: A rhesus macaque developed chronic anemia, lymphocytic leukocytopenia, fever, and anorexia while immunodeficient following inoculation with a simian-human immunodeficiency virus. METHODS: A complete blood count, peripheral blood smear, polymerase chain reaction and gene sequence were performed. RESULTS: Blood smears demonstrated persistent intraerythrocytic piroplasms with rare Maltese cross forms. Babesia microti-like protozoa were confirmed by polymerase chain reaction and sequencing of the 18S ribosomal RNA gene. CONCLUSION: With continued use of non-human primates as models for human diseases, infection and complications from babesiosis should be monitored.

Molecular characterization of Trypanosoma (Megatrypanum) spp. infecting cattle (Bos taurus), white-tailed deer (Odocoileus virginianus), and elk (Cervus elaphus canadensis) in the United States.

In the United States, the generally non-pathogenic trypanosome of cattle is designated Trypanosoma (Megatrypanum) theileri and is distinguished morphologically from Trypanosoma (M.) cervi, a trypanosome originally described in mule deer and elk. Phylogenetic studies of the Megatrypanum trypanosomes using various molecular markers reveal two lineages, designated TthI and TthII, with several genotypes within each. However, to date there is very limited genetic data for T. theileri, and none for the Megatrypanum trypanosomes found in wild ungulates, in the U.S. In this study U.S. isolates from cattle (Bos taurus), white-tailed deer (Odocoileus virginianus) (WTD), and elk (Cervus elaphus canadensis) were compared by ribosomal DNA (rDNA) sequence analysis and their incidence in cattle and WTD in south Texas counties was investigated. Phylogenetic analyses showed clear separation of the bovine and cervine trypanosomes. Both lineages I and II were represented in the U.S. cattle and WTD parasites. Lineage I cattle isolates were of a previously described genotype, whereas WTD and elk isolates were of two new genotypes distinct from the cattle trypanosomes. The cattle isolate of lineage II was of a previously reported genotype and was divergent from the WTD isolate, which was of a new genotype. In La Salle, Starr, Webb, and Zapata counties in south Texas a total of 51.8% of white-tailed deer were positive for trypanosomes by 18S rDNA PCR. Of the cattle screened in Webb County, 35.4% were positive. Drought conditions prevailing in south Texas when the animals were screened suggest the possibility of a vector for Trypanosoma other than the ked (Lipoptena mazamae) and tabanid flies (Tabanus spp. and Haematopota spp.).

Evaluation of methods for the isolation of high quality RNA from bovine and cervine hide biopsies.

Molecular investigations of the ruminant response to ectoparasites at the parasite-host interface are critically dependent upon the quality of RNA. The complexity of ruminant skin decreases the capacity to obtain high quality RNA from biopsy samples, which directly affects the reliability of data produced by gene expression experiments. Two methods for isolating total RNA from skin were compared and the use of 4M guanidinium isothiocyanate (GITC) during frozen storage of the specimens was evaluated. In addition, the best procedure for RNA isolation from bovine skin punch biopsies was also tested on white-tailed deer skin biopsies. Skin biopsy punches were collected and frozen prior to pulverization for RNA isolation. Total RNA quantity and integrity were determined by spectrophotometry and capillary electrophoresis technology, respectively. Significantly increased total RNA yield (P < 0.05) and higher integrity (P < 0.05) were obtained with a TRI Reagent® isolation method. Freezing and subsequent storage of bovine skin punch biopsies in 4 M GITC did not affect the amount or integrity of total RNA recovered by either RNA isolation method. However, quantity and integrity of total RNA extracted with the TRI Reagent method were again significantly higher than with the alternate technique, confirming it as the superior method. The TRI Reagent isolation method also yielded high quality total RNA from white-tailed deer skin punch biopsies, suggesting the usefulness of this method for obtaining RNA of a quality suitable for gene expression studies in other ruminant species.

Identification of a Mycoplasma ovis-like organism in a herd of farmed white-tailed deer (Odocoileus virginianus) in rural Indiana.

Mycoplasma ovis is a hemoplasma parasite of sheep, goats, and reindeer; however, natural hemoplasma infection in white-tailed deer has not previously been reported. Subsequent to finding many coccoid, bacillary, and ring-shaped organisms, consistent with hemotropic mycoplasmas, on RBCs from a 72-day-old female white-tailed fawn, we sought to (1) identify the putative hemoplasma observed in blood from the fawn, (2) evaluate others in the herd for hemoplasma infection, and (3) identify clinicopathologic characteristics of hemoplasma-infected white-tailed deer. EDTA-anticoagulated whole blood was collected from the fawn and 8 apparently healthy does in the same herd. CBCs were performed on 7 nonclotted samples from the fawn and 6 does. DNA was extracted from all samples, followed by PCR amplification of bacterial (16S rDNA) and protozoal (18S rDNA) genes. The nearly complete 16S rDNA product from the fawn's sample was directly sequenced and compared with known sequences in the GenBank database. Samples from the fawn and 7 of 8 does were PCR-positive using hemoplasma-specific and M ovis-specific protocols. The fawn was PCR-negative for Anaplasma spp., Babesia spp., and Theileria spp. The 16S rDNA sequence from the fawn (GenBank accession number, FJ824847) was most closely related to M ovis (AF338268), having 98.5% sequence identity. The fawn had a mild nonregenerative anemia, a neutrophilic left-shift with toxic change, aspiration bronchopneumonia, and gastrointestinal disease. Hematologic values, including blood film evaluation, in infected does were unremarkable. The M ovis-like organism may have acted as either an opportunistic or primary pathogen in the fawn. The high occurrence of subclinical infections in the does suggests that white-tailed deer may act as wildlife reservoirs for M ovis.

Molecular detection of Babesia bovis and Babesia bigemina in white-tailed deer (Odocoileus virginianus) from Tom Green County in central Texas.

Serologic and molecular evidence suggest that white-tailed deer in South Texas and North Mexico carry the agents of bovine babesiosis, Babesia bovis and Babesia bigemina. To determine if white-tailed deer in central Texas, which is outside the known occurrence of the vector tick at this time, harbor these parasites, blood samples from free-ranging and captive white-tailed deer (Odocoileus virginianus) in Tom Green County were tested by polymerase chain reaction (PCR) assays for B. bovis and B. bigemina 18S rDNA. Of the 25 samples tested, three (12%) were positive by nested PCR for B. bovis. This identity was confirmed by sequence analysis of the cloned 18S rDNA PCR product. Further confirmation was made by sequence analysis of the rRNA internal transcribed spacer (ITS) 1, 5.8S rRNA gene, and ITS 2 genomic region in two (representing samples from two different ranches) of the B. bovis positive samples. Three samples were positive by B. bigemina nested PCR, but sequencing of the cloned products confirmed only one animal positive for B. bigemina; Theileria spp. DNA was amplified from the other two animal samples. In addition to Theileria spp., two genotypically unique Babesia species sequences were identified among the cloned sequences produced by the B. bigemina primers in one sample. Phylogenetic analysis showed no separation of the deer B. bovis or B. bigemina 18S rDNA, or deer B. bovis ITS region sequences from those of bovine origin. Clarification of the possible role of white-tailed deer as reservoir hosts in maintaining these important pathogens of cattle is critical to understanding whether or not deer contribute to the epidemiology of bovine babesiosis.

Molecular and serologic evidence for Babesia bovis-like parasites in white-tailed deer (Odocoileus virginianus) in south Texas.

The current study was undertaken to determine if white-tailed deer in south Texas harbor Babesia bovis, a causative agent of bovine babesiosis. Blood samples from free-ranging white-tailed deer (Odocoileus virginianus) on two ranches in LaSalle and Webb Counties were screened for B. bovis and other hemoparasites by the polymerase chain reaction (PCR) to detect the piroplasm 18S rDNA. Serology was conducted on selected samples to detect antibody activity to B. bovis by the immunofluorescent antibody test (IFAT). PCR revealed that 16% of the LaSalle County samples and 4% of the Webb County samples were positive for B. bovis. Five of the LaSalle County and the two Webb County B. bovis 18S rDNA amplicons were cloned and sequenced. The resulting clones shared 99% identity to B. bovis 18S rRNA gene sequences derived from cattle isolates. Weak seroreactivity to B. bovis was shown by the IFAT. The samples were also screened for additional hemoparasites of deer including Theileria cervi, Babesia odocoilei and other Babesia spp. A genotypically unique Theileria sp. was found, along with T. cervi and B. odocoilei. The finding of putative B. bovis in white-tailed deer necessitates further study to determine if deer may act as a transient host or even a reservoir of infection for B. bovis pathogenic to cattle.

Sarcocystis sp. encephalomyelitis in a cat.

A 5-month-old male neutered domestic shorthair cat was evaluated for spinal pain, ataxia, and anisocoria. Neuroanatomic localization indicated diffuse or multifocal central nervous system disease. On cerebrospinal fluid analysis, neutrophilic pleocytosis and intracellular protozoal merozoites were observed. The merozoites were oval, 2-4 microm in width and 4-6 microm in length, and had linear arrays of nuclear material concentrated at one pole. Serum was positive for Sarcocystis sp. antibodies and negative for Toxoplasma gondii antibodies. The organism was determined to be either Sarcocystis neurona or Sarcocystis dasypi based on sequence analysis of the internal transcribed spacer 1 ribosomal RNA genomic region. Clinical disease resolved following treatment with 3 different protocols for protozoal infection. This case is the first to demonstrate the antemortem diagnosis and survival of a domestic cat with Sarcocystis sp.-associated encephalomyelitis. Clinicians and cytopathologists should include Sarcocystis sp. as a differential for feline inflammatory central nervous system disease characterized by neutrophilic pleocytosis.

Canine hepatozoonosis and babesiosis, and feline cytauxzoonosis.

The apicomplexan protozoans of the genera Hepatozoon, Babesia, and Cytauxzoon are emerging parasite pathogens that are increasingly diagnosed in the pet population. These tick-transmitted apicomplexan parasites are becoming more widely recognized as serious canine or feline pathogens. This article reviews the epidemiology, diagnosis, treatment, and control of canine hepatozoonosis and babesiosis, and feline cytauxzoonosis.