ABSTRACT
In 2020, Rickettsia typhi was diagnosed in a dog from Houston, Texas, USA based upon R. typhi IFA seroreactivity in both acute and convalescent sera, and PCR with DNA sequencing of 4 different gene regions, all of which were 100% identical to R. typhi. The dog was clinically ill with intermittent fever, lethargy, inappetence, and lymphadenopathy. Clinicopathological abnormalities included a mild nonregenerative anemia, neutrophilia, lymphopenia, thrombocytopenia, hypoalbuminemia, and elevated ALP. The dog rapidly recovered with doxycycline administration.
Subject(s)
Dog Diseases , Typhus, Endemic Flea-Borne , Animals , Dog Diseases/diagnosis , Dog Diseases/drug therapy , Dogs , Doxycycline/therapeutic use , Polymerase Chain Reaction/veterinary , Rickettsia typhi , Texas , Typhus, Endemic Flea-Borne/diagnosis , Typhus, Endemic Flea-Borne/drug therapy , Typhus, Endemic Flea-Borne/veterinaryABSTRACT
Accurate detection of vector-borne pathogens (VBPs) is extremely important as the number of reported cases in humans and animals continues to rise in the US and abroad. Validated PCR assays are currently the cornerstone of molecular diagnostics and can achieve excellent analytical sensitivity and specificity. However, the detection of pathogens at low parasitemia still presents a challenge for VBP diagnosis, especially given the very low volume of specimens tested by molecular methods. The objective of this study is to determine if a commercially available microbial enrichment kit, used prior DNA extraction, is capable of expanding the overall microbial community and increasing detectable levels of VBPs in canine blood samples through host DNA depletion. This study used EDTA-whole blood samples from dogs naturally infected with varying parasitemia levels of either Anaplasma phagocytophilum, Babesia gibsoni, or Ehrlichia ewingii. For two VBPs, EDTA-blood samples were diluted to determine the effect of microbial concentration at low parasitemia. Paired EDTA-blood samples from each dog were subjected to traditional, automated DNA extraction with or without the microbial concentrating kit (MolYsis®) prior DNA extraction. Relative amounts of pathogen DNA in paired samples were determined by real-time PCR and Next-Generation Sequencing targeting conserved regions of 16S rRNA (for bacteria) and 18S rRNA (for protozoa). Results from the three molecular methods suggest that the microbial concentrating kit did not improve the detection of VBPs, although significantly reduced the presence of host DNA. Alternative methods for VBP enrichment in clinical samples prior to molecular testing should continue to be investigated, as it may significantly improve clinical sensitivity and reduce the number of false-negative results.
Subject(s)
DNA, Bacterial/isolation & purification , DNA, Protozoan/isolation & purification , Dog Diseases/diagnosis , Vector Borne Diseases/diagnosis , Anaplasma/genetics , Anaplasma phagocytophilum , Animals , Bacteria/genetics , Dogs , Ehrlichia/genetics , High-Throughput Nucleotide Sequencing , Microbiota , Polymerase Chain Reaction , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 18S/genetics , Real-Time Polymerase Chain Reaction , Tick-Borne Diseases , Vector Borne Diseases/microbiology , Vector Borne Diseases/parasitologyABSTRACT
In 2018 and 2019, spotted fever was suspected in 3 dogs in 3 US states. The dogs had fever and hematological abnormalities; blood samples were Rickettsia seroreactive. Identical Rickettsia DNA sequences were amplified from the samples. Multilocus phylogenetic analysis showed the dogs were infected with a novel Rickettsia species related to human Rickettsia pathogens.
