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Anaplasma spp., Ehrlichia spp., Rickettsia spp., and Lyme disease associated Borrelia spp. are the most common tick-borne pathogens reported to infect human beings worldwide and other animals, such as dogs and horses. In the present study, we developed a broad-coverage SYBR Green QPCR panel consisting of four individual assays for the detection and partial differentiation of the aforementioned pathogens. All assays were optimized to the same thermocycling condition and had a detection limit of 10 copies per reaction. The assays remained sensitive when used to test canine and equine blood DNA samples spiked with known amounts of synthetic DNA (gBlock) control template. The assays were specific, as evidenced by lack of cross reaction to non-target gBlock or other pathogens commonly tested in veterinary diagnostic labs. With appropriate Ct cutoff values for positive samples and negative controls and the melting temperature (TM) ranges established in the present study, the QPCR panel is suitable for accurate, convenient and rapid screening and confirmation of tick-borne pathogens in animals., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Dogs in the United States are hosts to a diverse range of ticks and tick-borne pathogens, including A. phagocytophilum, an important emerging canine and human pathogen. Previously, a Companion Animal Parasite Council (CAPC)-sponsored workshop proposed factors purported to be associated with the infection risk for tick-transmitted pathogens in dogs in the United States, including climate conditions, socioeconomic characteristics, local topography, and vector distribution., Methods: Approximately four million test results from routine veterinary diagnostic tests from 2011-2013, which were collected on a county level across the contiguous United States, are statistically analyzed with the proposed factors via logistic regression and generalized estimating equations. Spatial prevalence maps of baseline Anaplasma spp. prevalence are constructed from Kriging and head-banging smoothing methods., Results: All of the examined factors, with the exception of surface water coverage, were significantly associated with Anaplasma spp. prevalence. Overall, Anaplasma spp. prevalence increases with increasing precipitation and forestation coverage and decreases with increasing temperature, population density, relative humidity, and elevation. Interestingly, socioeconomic status and deer/vehicle collisions were positively and negatively correlated with canine Anaplasma seroprevalence, respectively. A spatial map of the canine Anaplasma hazard is an auxiliary product of the analysis. Anaplasma spp. prevalence is highest in New England and the Upper Midwest., Conclusions: The results from the two posited statistical models (one that contains an endemic areas assumption and one that does not) are in general agreement, with the major difference being that the endemic areas model estimates a larger prevalence in Western Texas, New Mexico, and Colorado. As A. phagocytophilum is zoonotic, the results of this analysis could also help predict areas of high risk for human exposure to this pathogen.

Canine monocytic ehrlichiosis caused by Ehrlichia canis is of veterinary importance worldwide. In Thailand, there has been little information available on E. canis and its phylogeny. The objective of this study was to characterize and establish molecular structure and phylogeny of Thai Ehrlichia and Anaplasma strains. Genus-specific primers for Ehrlichia and Anaplasma were used to amplify the 16S rRNA gene from naturally infected canine blood samples, and these amplicon sequences were compared with other sequences from GenBank. Both homology and secondary structure analysis of 16S rRNA sequences indicated that they were novel E. canis and A. platys strains. Phylogenetic analysis revealed that the Thai E. canis strain was closely related and formed a single cluster with E. canis from different countries. A. platys found in this study showed close relationship with earlier report of A. platys from Thailand. To our knowledge this report represents the first molecular characterization of the nearly complete 16S rRNA gene from E. canis in dogs from Thailand.

Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne zoonosis caused by a strain of Anaplasma phagocytophila called the HGE agent, an obligatory intracellular bacterium. The agent expresses immunodominant 44-kDa outer membrane proteins (P44s) encoded by a multigene family. The present study established an experimental process for transmission of the HGE agent from infected mice (a reservoir model) to nymphal Ixodes scapularis ticks (a biological vector) and subsequently to horses (a patient model) by the adult infected ticks. Overall, a total of 20 different p44 transcripts were detected in the mammals, ticks, and cell cultures. Among them, a transcript from a p44-18 gene was major at acute stage in mice and horses but minor in ticks. Both mRNA and protein produced from the p44-18 gene were detected in the HGE agent cultivated in HL-60 cells at 37 degrees C, but their expression levels decreased in the organisms cultivated at 24 degrees C, suggesting that temperature is one of the factors that influence the expression of members of the p44 multigene family. Several additional p44 transcripts that were not detected in the mammals at the acute stage of infection were detected in ticks. Phylogenetic analysis of the 20 different p44 transcripts revealed that the major transcripts found in mammals and ticks were distinct, suggesting a difference in surface properties between populations of the HGE agent in different host environments. The present study provides new information for understanding the role of the p44 multigene family in transmission of the HGE agent between mammals and ticks.

The polymerase chain reaction (PCR) was used to detect Anaplasma marginale in hemolymph collected from live Dermacentor andersoni Stiles ticks. Hemolymph was collected from severed legs of male and female ticks exposed to A. marginale as either nymphs or adults. Heat treatment was found to be the optimum method of hemolymph preparation for PCR. Hemolymph samples were collected and pooled from adult ticks exposed as nymphs on days 0-10 of feeding on a susceptible calf. For male and female ticks exposed as adults, samples were collected as ticks fed 7 d on an infected calf, while being held 9 d between feedings, and during a second feeding of 10 d (or to repletion) when they transmitted the parasite. Hemolymph samples were collected from uninfected ticks at the same times to serve as controls. Anaplasma marginale DNA was amplified with primers BAP-2 (5'-GTATGGCACGTAGTCTTGGGATCA-3') and AL34S (5'-CAGCAGCAGCAAGACCTTCA-3'), which flank a 409-bp fragment of the A. marginale Florida isolate msp1 beta gene. Infected tick hemolymph was PCR-positive for A. marginale at all collection times, including unfed adults infected as nymphs and previously unexposed adults that fed on infected calves for only 1 d. The PCR-based assay of tick hemolymph proved to be a sensitive method for identification of infected ticks, potentially without killing them; it would be well suited for identification of laboratory- or field-infected ticks that could then be used for further studies. The primers used in this assay were also found specific when tested with species of 18 different genera, and universal for 7 A. marginale isolates from diverse geographical areas of the United States.

The polymerase chain reaction (PCR) was used to detect Anaplasma marginale in secretagogue-induced oral secretions of male and female Dermacentor andersoni Stiles exposed as nymphs or adults by feeding on infected calves. A 409-bp DNA fragment derived from the A. marginale (Florida isolate) msp1 beta gene was amplified with oligonucleotide primers BAP-2 (5'-GTATGGCACGTAGTCTTGGGATCA-3') and AL34S (5'-CAGCAGCAGCAAGACCTTCA-3'). The target DNA was amplified in oral secretions of female ticks exposed to A. marginale as adults and stimulated to secrete by injection of dopamine. Conversely, A. marginale was detected in saliva from prefed female ticks exposed as nymphs only after stimulation with a combination of dopamine, gamma-aminobutyric acid, pilocarpine, and theophylline. Saliva from ticks exposed as nymphs and stimulated with ergot alkaloids did not contain the A. marginale target DNA. Saliva collected after 11 d of feeding from dopamine-stimulated male ticks contained A. marginale DNA. The results indicate that A. marginale is present in tick saliva and suggest that the parasite can be transmitted to cattle via saliva of feeding ixodid ticks. The variable appearance of A. marginale in saliva, regardless of the method used to induce salivation, suggests that transmission of A. marginale may be affected by the physiological state of the tick.

The development of Anaplasma marginale in midgut epithelial cells was studied in feeding, transmitting adult Dermacentor andersoni ticks. Laboratory-reared ticks experimentally infected as nymphs were allowed to feed from 1 to 9 days on susceptible calves. Gut tissues from ticks were collected on each day they fed (total, 9 days) and were processed for light and transmission electron microscopy. Colonies of A marginale were abundant during the first 6 days of feeding, after which numbers decreased. Colonies were adherent to the basement membrane of gut cells early during feeding, with resultant flattening of the colonies. Colonies also were seen in muscle cells on the hemocoel side of the basement membrane. Morphologic features of A marginale within muscle cells varied and were similar to those observed in gut cells. In addition, however, a large reticulated form in the colonies was observed in muscle cells and appeared to give rise to small particles by budding. Development of A marginale in muscle cells appears to represent an intermediate site of development between those in gut and in salivary glands.

Transstadial and transovarial transmission of Anaplasma marginale by Dermacentor variabilis were attempted with with ticks exposed to the organism once by feeding as larvae or nymphs, and twice by feeding as larvae and nymphs. Typical colonies of A marginale were in gut tissues of adults that were infected as larvae, larvae and nymphs, and as nymphs; repeated exposure of ticks did not appear to result in an increase in the number of colonies in the gut of subsequently molted adults nor did it affect severity of the clinical disease that developed in cattle they fed on. In contrast, colonies of A marginale were not found in the midgut epithelium of unfed nymphs exposed as larvae, even though companion nymphs transmitted the parasite, causing severe clinical anaplasmosis in susceptible calves. The organism was not transmitted transovarially by F1 larvae or nymphs from the groups exposed as parent larvae, nymphs, larvae and nymphs, and as adults. Some of the calves fed on by F1 progeny had a few erythrocytic marginale bodies that looked suspiciously like A marginale, as well as postchallenge exposure prepatent periods that were longer than other calves in the transovarial transmission study. Sera from these calves were tested for antibody to A marginale, using a highly sensitive immunoblot technique. Antibodies were not detected in any of the sera.