Comparative genomics of Giardia duodenalis sub-assemblage AI beaver (Be-2) and human (WB-C6) strains show remarkable homozygosity, sequence similarity, and conservation of VSP genes.

Giardia duodenalis, a major cause of waterborne infection, infects a wide range of mammalian hosts and is subdivided into eight genetically well-defined assemblages named A through H. However, fragmented genomes and a lack of comparative analysis within and between the assemblages render unclear the molecular mechanisms controlling host specificity and differential disease outcomes. To address this, we generated a near-complete de novo genome of AI assemblage using the Oxford Nanopore platform by sequencing the Be-2 genome. We generated 148,144 long-reads with quality scores of > 7. The final genome assembly consists of only nine contigs with an N50 of 3,045,186 bp. This assembly agrees closely with the assembly of another strain in the AI assemblage (WB-C6). However, a critical difference is that a region previously placed in the five-prime region of Chr5 belongs to Chr4 of Be-2. We find a high degree of conservation in the ploidy, homozygosity, and the presence of cysteine-rich variant-specific surface proteins (VSPs) within the AI assemblage. Our assembly provides a nearly complete genome of a member of the AI assemblage of G. duodenalis, aiding population genomic studies capable of elucidating Giardia transmission, host range, and pathogenicity.

Trichinella: Becoming a parasite.

Phylogenetic evidence indicates that free-living nematodes gave rise to parasitic nematodes where parasitism evolved independently at least 15 times. The high level of genetic and biological diversity among parasites dictates an equally high level of diversity in the transition to parasitism. We previously hypothesized that horizontal gene transfer (HGT) played an important role in the evolution of parasitism among early ancestors of Trichinella, mediated by an interplay of ecological and evolutionary pathways that contributed to persistence and diversification. We propose that host selection may have been associated with the metabolism of ammonia and engender a new paradigm whereby the reprogrammed nurse cell is capable of generating cyanate thereby enabling the importance of the Trichinella cyanase in the longevity of the cell. Parasites and parasitism have revealed considerable resilience against a backdrop of climate change and environmental perturbation. Here we provide a putative link between key periods in the evolution of Trichinella and major geological and climatological events dating back 500 million years. A useful lens for exploring such ideas, the Stockholm Paradigm, integrates Ecological Fitting (a foundation for host colonization and diversification), the Oscillation Hypothesis (recurring shifts between trends in generalization and specialization relative to host range), the Geographic Mosaic Theory of Coevolution (microevolutionary co-adaptive processes), and the Taxon Pulse Hypothesis (alternating events of biotic expansion i.e., exploitation in evolutionary and ecological time). Here we examine how one or more of these interactive theories, in a phylogenetic-historical context and in conjunction with HGT, may help explain the scope and depth of diversity among Trichinella genotypes.

Aqueous Ozone Exposure Inhibits Sporulation in the Cyclospora cayetanensis Surrogate Eimeria acervulina.

Ozone is a potent disinfecting agent used to treat potable water and wastewater, effectively clearing protozoa such as Giardia and Cryptosporidium spp. It is unclear whether ozone treatment of water or fresh produce can reduce the spread of the emerging parasite Cyclospora cayetanensis, which causes cyclosporiasis in humans. Obtaining viable C. cayetanensis oocysts to evaluate inactivation methods is challenging because we lack the means to propagate them in vitro, because of delays in case reporting, and because health departments typically add inactivating fixatives to clinical specimens. Research in various surrogate organisms has sought to bolster understanding of the biology of C. cayetanensis. Among these surrogates is the poultry parasite Eimeria acervulina, a closely related and easily cultured parasite of economic significance. We used this surrogate to evaluate the consequences of ozone treatment, using the sporulation state as an indicator of infectious potential. Treating with ozonated water acidified with citric acid reduced sporulation ability in a dose-dependent manner; treatment with up to 4.93 mg/L initial concentration of ozone resulted in a 93% inactivation of sporulation by 7 days posttreatment. This developmental arrest was accompanied by transcriptional changes in genes involved in regulating the response to reactive oxygen species (ROS) in a time course that is consistent with the production of oxygen free radicals. This study shows that ozone is highly effective in preventing sporulation of E. acervulina, a model coccidian used as a surrogate for Cyclospora. Furthermore, ozone exposure induced molecular responses to general oxidative stress, documented with several well-characterized antioxidant enzymes.

Living Donor Liver Transplantation for Adults With High Model for End-stage Liver Disease Score: The US Experience.

BACKGROUND: Outcomes after living-donor liver transplantation (LDLT) at high Model for End-stage Liver Disease (MELD) scores are not well characterized in the United States. METHODS: This was a retrospective cohort study using Organ Procurement and Transplantation Network data in adults listed for their first liver transplant alone between 2002 and 2021. Cox proportional hazards models evaluated the association of MELD score (<20, 20-24, 25-29, and ≥30) and patient/graft survival after LDLT and the association of donor type (living versus deceased) on outcomes stratified by MELD. RESULTS: There were 4495 LDLTs included with 5.9% at MELD 25-29 and 1.9% at MELD ≥30. LDLTs at MELD 25-29 and ≥30 LDLT have substantially increased since 2010 and 2015, respectively. Patient survival at MELD ≥30 was not different versus MELD <20: adjusted hazard ratio 1.67 (95% confidence interval, 0.96-2.88). However, graft survival was worse: adjusted hazard ratio (aHR) 1.69 (95% confidence interval, 1.07-2.68). Compared with deceased-donor liver transplant, LDLT led to superior patient survival at MELD <20 (aHR 0.92; P = 0.024) and 20-24 (aHR 0.70; P < 0.001), equivalent patient survival at MELD 25-29 (aHR 0.97; P = 0.843), but worse graft survival at MELD ≥30 (aHR 1.68, P = 0.009). CONCLUSIONS: Although patient survival remains acceptable, the benefits of LDLT may be lost at MELD ≥30.

Chromosomal scale assembly reveals localized structural variants in avian caecal coccidian parasite Eimeria tenella.

Eimeria tenella is a major cause of caecal coccidiosis in commercial poultry chickens worldwide. Here, we report chromosomal scale assembly of Eimeria tenella strain APU2, a strain isolated from commercial broiler chickens in the U.S. We obtained 100× sequencing Oxford Nanopore Technology (ONT) and more than 800× Coverage of Illumina Next-Seq. We created the assembly using the hybrid approach implemented in MaSuRCA, achieving a contiguous 51.34 Mb chromosomal-scale scaffolding enabling identification of structural variations. The AUGUSTUS pipeline predicted 8060 genes, and BUSCO deemed the genomes 99% complete; 6278 (78%) genes were annotated with Pfam domains, and 1395 genes were assigned GO-terms. Comparing E. tenella strains (APU2, US isolate and Houghton, UK isolate) derived Houghton strain of E. tenella revealed 62,905 high stringency differences, of which 45,322 are single nucleotide polymorphisms (SNPs) (0.088%). The rate of transitions/transversions among the SNPs are 1.63 ts/tv. The strains possess conserved gene order but have profound sequence heterogeneity in a several chromosomal segments (chr 2, 11 and 15). Genic and intergenic variation in defined gene families was evaluated between the two strains to possibly identify sequences under selection. The average genic nucleotide diversity of 2.8 with average 2 kb gene length (0.145%) at genic level. We examined population structure using available E. tenella sequences in NCBI, revealing that the two E. tenella isolates from the U.S. (E. tenella APU2 and Wisconsin, "ERR296879") share a common maternal inheritance with the E. tenella Houghton. Our chromosomal level assembly promotes insight into Eimeria biology and evolution, hastening drug discovery and vaccine development.

Sarcocystis cruzi (Hasselmann, 1923) Wenyon, 1926: redescription, molecular characterization and deposition of life cycle stages specimens in the Smithsonian Museum.

Currently, 7 named Sarcocystis species infect cattle: Sarcocystis hirsuta, S. cruzi, S. hominis, S. bovifelis, S. heydorni, S. bovini and S. rommeli; other, unnamed species also infect cattle. Of these parasites of cattle, a complete life cycle description is known only for S. cruzi, the most pathogenic species in cattle. The life cycle of S. cruzi was completed experimentally in 1982, before related parasite species were structurally characterized, and before the advent of molecular diagnostics; to our knowledge, no archived frozen tissues from the cattle employed in the original descriptions remain for DNA characterization. Here, we isolated DNA from a paraffin-embedded kidney of a calf experimentally infected with S. cruzi in 1980; we then sequenced portions of 18S rRNA, 28S rRNA, COX1 and Acetyl CoA genes and verified that each shares 99­100% similarity to other available isolates attributed to S. cruzi from naturally infected cattle. We also reevaluated histological sections of tissues of calves experimentally infected with S. cruzi in the original description, exploiting improvements in photographic technology to render clearer morphological detail. Finally, we reviewed all available studies of the life cycle of S. cruzi, noting that S. cruzi was transmitted between bison (Bison bison) and cattle (Bos taurus) and that the strain of parasite derived from bison appeared more pathogenic than the cattle strain. Based on these newfound molecular, morphological and physiological data, we thereby redescribed S. cruzi and deposited reference material in the Smithsonian Museum for posterity.

The same genotype of Sarcocystis neurona responsible for mass mortality in marine mammals induced a clinical outbreak in raccoons (Procyon lotor) 10 years later.

Here, we report the first known outbreak of clinical protozoal myeloencephalitis in naturally infected raccoons by the parasite Sarcocystis neurona. The North American opossum (Didelphis virginiana) and the South American opossum (Didelphis albiventris) are its known definitive hosts. Several other animal species are its intermediate or aberrant hosts. The raccoon (Procyon lotor) is considered the most important intermediate host for S. neurona in the USA. More than 50% of raccoons in the USA have sarcocysts in their muscles, however clinical sarcocystosis in raccoons is rare. In 2014, 38 free-living raccoons were found dead or moribund on the grounds of the Saint Louis Zoo, Missouri, USA. Moribund individuals were weak, lethargic, and mildly ataxic; several with oculo-nasal discharge. Seven raccoons were found dead and 31 were humanely euthanized. Postmortem examinations were conducted on nine raccoons. Neural lesions compatible with acute sarcocystosis were detected in eight raccoons. The predominant lesions were meningoencephalitis and perivascular mononuclear cells. Histologic evidence for the Canine Distemper Virus was found in one raccoon. Schizonts and merozoites were present in the encephalitic lesions of four raccoons. Mature sarcocysts were present within myocytes of five raccoons. In six raccoons, S. neurona schizonts and merozoites were confirmed by immunohistochemical staining with S. neurona-specific polyclonal antibodies. Viable S. neurona was isolated from the brains of two raccoons by bioassay in interferon gamma gene knockout mice and in cell cultures seeded directly with raccoon brain homogenate. Molecular characterization was based on raccoon no. 68. Molecular characterization based on multi-locus typing at five surface antigens (SnSAG1-5-6, SnSAG3 and SnSAG4) and the ITS-1 marker within the ssrRNA locus, using DNA isolated from bradyzoites released from sarcocysts in a naturally infected raccoon (no. 68), confirmed the presence of S. neurona antigen type I, the same genotype that caused a mass mortality event in which 40 southern sea otters stranded dead or dying within a 3 week period in April 2004 with S. neurona-associated disease. An expanded set of genotyping markers was next applied. This study reports the following new genotyping markers at 18S rRNA, 28S rRNA, COX1, ITS-1, RON1, RON2, GAPDH1, ROP20, SAG2, SnSRS21 and TUBA1 markers. The identity of Sarcocystis spp. infecting raccoons is discussed.

Genetic diversity of Plasmodium vivax populations from the China-Myanmar border identified by genotyping merozoite surface protein markers.

BACKGROUND: Parasite diversity and population structure influence malaria control measures. Malaria transmission at international borders affects indigenous residents and migrants, defying management efforts and resulting in malaria re-introduction. Here we aimed to determine the extent and distribution of genetic variations in Plasmodium vivax populations and the complexity of infections along the China-Myanmar border. METHODS: We collected clinical P. vivax samples from local and migrant malaria patients from Laiza and Myitsone, Kachin State, Myanmar, respectively. We characterized the polymorphisms in two P. vivax merozoite surface protein markers, Pvmsp-3α and Pvmsp-3ß, by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. We sought to determine whether these genetic markers could differentiate these two neighboring parasite populations. RESULTS: PCR revealed three major size variants for Pvmsp-3α and four for Pvmsp-3ß among the 370 and 378 samples, respectively. PCR-RFLP resolved 26 fragment-size alleles by digesting Pvmsp-3α with Alu I and Hha I and 28 alleles by digesting Pvmsp-3ß with Pst I. PCR-RFLP analysis of Pvmsp-3α found that infections in migrant laborers from Myitsone bore more alleles than did infections in residents of Laiza, while such difference was not evident from genotyping Pvmsp-3ß. Infections originating from these two places contained distinct but overlapping subpopulations of P. vivax. Infections from Myitsone had a higher multiplicity of infection as judged by the size of the Pvmsp-3α amplicons and alleles after Alu I/Hha I digestion. CONCLUSIONS: Migrant laborers from Myitsone and indigenous residents from Laiza harbored overlapping but genetically distinct P. vivax parasite populations. The results suggested a more diverse P. vivax population in Myitsone than in the border town of Laiza. PCR-RFLP of Pvmsp-3α offers a convenient method to determine the complexity of P. vivax infections and differentiate parasite populations.

RNA-Seq of Phenotypically Distinct Eimeria maxima Strains Reveals Coordinated and Contrasting Maturation and Shared Sporogonic Biomarkers with Eimeria acervulina.

Strains of Eimeria maxima, an enteric parasite of poultry, vary in virulence. Here, we performed microscopy and RNA sequencing on oocysts of strains APU-1 (which exhibits more virulence) and APU-2. Although each underwent parallel development, APU-1 initially approached maturation more slowly. Each strain sporulated by hour 36; their gene expression diverged somewhat thereafter. Candidate biomarkers of viability included 58 genes contributing at least 1000 Transcripts Per Million throughout sporulation, such as cation-transporting ATPases and zinc finger domain-containing proteins. Many genes resemble constitutively expressed genes also important to Eimeria acervulina. Throughout sporulation, the expression of only a few genes differed between strains; these included cyclophilin A, EF-1α, and surface antigens (SAGs). Mature and immature oocysts uniquely differentially express certain genes, such as an X-Pro dipeptidyl-peptidase domain-containing protein in immature oocysts and a profilin in mature oocysts. The immature oocysts of each strain expressed more phosphoserine aminotransferase and the mature oocysts expressed more SAGs and microneme proteins. These data illuminate processes influencing sporulation in Eimeria and related genera, such as Cyclospora, and identify biological processes which may differentiate them. Drivers of development and senescence may provide tools to assess the viability of oocysts, which would greatly benefit the poultry industry and food safety applications.

PvMSP-3α and PvMSP-3ß genotyping reveals higher genetic diversity in Plasmodium vivax parasites from migrant workers than residents at the China-Myanmar border.

BACKGROUND: The genetic diversity of malaria parasites traces the origin and spread of new variants and can be used to evaluate the effectiveness of malaria control measures. Therefore, this study aims to improve the understanding of the molecular epidemiology of Plasmodium vivax malaria at the China-Myanmar border by genotyping the PvMSP-3α and PvMSP-3ß genes. METHODS: Blood samples were collected from P. vivax malaria patients along the China-Myanmar border. The PvMSP-3α and PvMSP-3ß genes were amplified by polymerase chain reaction (PCR) and the genetic polymorphism and haplotype of the two genes were analyzed. RESULTS: A total of 422 blood samples were used for this study, of which 224 were analyzed at PvMSP-3α and 126 at PvMSP-3ß. Samples mainly were from young adults aged 18-45 years, although local patients were significantly younger than migrant laborers crossing the border at Tengchong (P < 0.0001). Molecular evolutionary analysis revealed that PvMSP-3α and PvMSP-3ß underwent diversifying natural selection, and intragenic recombination contributed to the diversity of the isolates. Based on the length of the genes, we identified three types of PvMSP-3α [1.9-2.0 kb (Type-A), 1.4-1.5 kb (Type-B), and 1.1-1.3 kb (Type-C)] and two types of PvMSP-3ß [1.7-2.2 kb (Type-A) and 1.4-1.5 kb (Type-B)]. Migrant laborers returning to China through Tengchong bore P. vivax infections displaying significantly higher genetic diversity than local residents. CONCLUSIONS: Both PvMSP-3 paralogs were subjected to diversifying selection in each sample population. Clustering of alleles supports ephemeral endemic differentiation of alleles, but the broader phylogeny suggests that alleles transit the globe, perhaps accelerated by movements of migrants such as those transiting Tengchong.

Hastening Progress in Cyclospora Requires Studying Eimeria Surrogates.

Cyclospora cayetanensis is an enigmatic human parasite that sickens thousands of people worldwide. The scarcity of research material and lack of any animal model or cell culture system slows research, denying the produce industry, epidemiologists, and regulatory agencies of tools that might aid diagnosis, risk assessment, and risk abatement. Fortunately, related species offer a strong foundation when used as surrogates to study parasites of this type. Species of Eimeria lend themselves especially well as surrogates for C. cayetanensis. Those Eimeria that infect poultry can be produced in abundance, share many biological features with Cyclospora, pose no risk to the health of researchers, and can be studied in their natural hosts. Here, we overview the actual and potential uses of such surrogates to advance understanding of C. cayetanensis biology, diagnostics, control, and genomics, focusing on opportunities to improve prevention, surveillance, risk assessment, and risk reduction. Studying Eimeria surrogates accelerates progress, closing important research gaps and refining promising tools for producers and food safety regulators to monitor and ameliorate the food safety risks imposed by this emerging, enigmatic parasite.

Life Cycle and Transmission of Cyclospora cayetanensis: Knowns and Unknowns.

Although infections with Cyclospora cayetanensis are prevalent worldwide, many aspects of this parasite's life cycle and transmission remain unknown. Humans are the only known hosts of this parasite. Existing information on its endogenous development has been derived from histological examination of only a few biopsy specimens. Its asexual and sexual stages occur in biliary-intestinal epithelium. In histological sections, its stages are less than 10 µm, making definitive identification difficult. Asexual (schizonts) and sexual (gamonts) are located in epithelial cells. Male microgamonts have two flagella; female macrogametes contain wall-forming bodies. Oocysts are excreted in feces unsporulated. Sporulation occurs in the environment, but there are many unanswered questions concerning dissemination and survival of C. cayetanensis oocysts. Biologically and phylogenetically, C. cayetanensis closely resembles Eimeria spp. that parastize chickens; among them, E. acervulina most closely resembles C. cayetanensis in size. Here, we review known and unknown aspects of its life cycle and transmission and discuss the appropriateness of surrogates best capable of hastening progress in understanding its biology and developing mitigating strategies.

Trich-tracker - a practical tool to trace Trichinella spiralis transmission based on rapid, cost-effective sampling of genome-wide genetic variation.

Molecular epidemiology using traditional sequencing has been notoriously difficult in inbred parasites due to a lack of genetic variation available for discriminating among parasites. Next generation sequencing techniques offer a solution to this problem by increasing the number of loci that can be sequenced. Here, we introduce Trich-tracker, a tool that makes efficient use of diagnostic variation distributed throughout the genome of Trichinella spiralis to more rapidly, and conclusively, resolve connections and distinctions among focal outbreaks of T. spiralis. In particular, we rapidly characterised genetic variation among a sample of parasites from Polish farms and wildlife, sampling genomic variation using double digest restriction site-associated DNA sequencing (ddRADseq). Approximately 400,000 bases of sequence were generated from each sample and shown to be distributed across the genome with single nucleotide polymorphisms occurring at a frequency of approximately one base in 10,000. Both phylogenetic and Bayesian clustering analyses indicated that ddRADseq genotypes formed distinct clusters for specific outbreaks and were quite distinct from wild boar samples. Two of the investigated outbreaks were more similar to each other than to other outbreak samples, suggesting a link between these outbreaks. Hence, the Trich-tracker procedure identified informative genomic variation which afforded unprecedented epidemiological resolution. Trich-tracker is very flexible tool, quickly and inexpensively mining genomes of even highly inbred populations of T. spiralis to support outbreak investigations. The simplicity of the entire procedure, and time and cost effectiveness of Trich-tracker support its practical application in ongoing Trichinella outbreaks. The discriminating power of this tool is tunable and scalable, allowing application in a variety of epidemiological contexts, and is easily adapted to other parasite systems.

Comparison of in vitro transformation efficiency methods for Plasmodium falciparum.

Poor efficiency plagues conventional methods to transfect Plasmodium falciparum with genetic modifications, impeding research aimed at limiting the damage wrought by this agent of severe malaria. Here, we sought and documented improvements, using fluoresce imaging, cell sorting, and drug selection as means to measure efficiency. Through the transfection of EGFP plasmid, the transfection efficiency of the three methods used in this study was as high as 10-3. A method that pre-loaded uninfected erythrocytes with plasmids using the Bio-Rad Gene Pulser Xcell achieved the highest efficiency (0.48%±0.06%), twice the efficiency of a method using nuclear transfection of ring stages employing the 4D-NucleofectorTM X Kit L. We also evaluated an approach using the Nucleofactor system to transform schizont stages. We considered efficiency and the time required to complete drug screening experiments when evaluating transfection methods. Fluorescence measurements confirmed greater efficiencies for the Pre-load method (52.4% vs. 25%; P < 0.0001), but the Nuc-Ring method required less time to complete drug selection experiments following CRISPR/Cas9 editing. These data should benefit future studies seeking to remove or modify genes of P. falciparum.

Dynamically expressed genes provide candidate viability biomarkers in a model coccidian.

Eimeria parasites cause enteric disease in livestock and the closely related Cyclospora cayetanensis causes human disease. Oocysts of these coccidian parasites undergo maturation (sporulation) before becoming infectious. Here, we assessed transcription in maturing oocysts of Eimeria acervulina, a widespread chicken parasite, predicted gene functions, and determined which of these genes also occur in C. cayetanensis. RNA-Sequencing yielded ~2 billion paired-end reads, 92% of which mapped to the E. acervulina genome. The ~6,900 annotated genes underwent temporally-coordinated patterns of gene expression. Fifty-three genes each contributed >1,000 transcripts per million (TPM) throughout the study interval, including cation-transporting ATPases, an oocyst wall protein, a palmitoyltransferase, membrane proteins, and hypothetical proteins. These genes were enriched for 285 gene ontology (GO) terms and 13 genes were ascribed to 17 KEGG pathways, defining housekeeping processes and functions important throughout sporulation. Expression differed in mature and immature oocysts for 40% (2,928) of all genes; of these, nearly two-thirds (1,843) increased their expression over time. Eight genes expressed most in immature oocysts, encoding proteins promoting oocyst maturation and development, were assigned to 37 GO terms and 5 KEGG pathways. Fifty-six genes underwent significant upregulation in mature oocysts, each contributing at least 1,000 TPM. Of these, 40 were annotated by 215 GO assignments and 9 were associated with 18 KEGG pathways, encoding products involved in respiration, carbon fixation, energy utilization, invasion, motility, and stress and detoxification responses. Sporulation orchestrates coordinated changes in the expression of many genes, most especially those governing metabolic activity. Establishing the long-term fate of these transcripts in sporulated oocysts and in senescent and deceased oocysts will further elucidate the biology of coccidian development, and may provide tools to assay infectiousness of parasite cohorts. Moreover, because many of these genes have homologues in C. cayetanensis, they may prove useful as biomarkers for risk.

Infection, genetics, and evolution of Trichinella: Historical insights and applications to molecular epidemiology.

Genetic variation in pathogen populations provides the means to answer questions in disease ecology and transmission, illuminating interactions between genetic traits, environmental exposures, and disease. Such studies elucidate the phylogeny, evolution, transmission and pathogenesis of viruses, bacteria and parasites. Here, we review how such studies have fostered understanding of the biology and epidemiology of zoonotic nematode parasites in the genus Trichinella spp., which impose considerable economic and health burdens by infecting wildlife, livestock, and people. To use such data to define ongoing chains of local transmission and source traceback, researchers first must understand the extent and distribution of genetic variation resident in regional parasite populations. Thus, genetic variability illuminates a population's past as well as its present. Here we review how such data have helped define population dynamics of Trichinella spp. in wild and domesticated hosts, creating opportunities to harness genetic variation in the quest to prevent, track, and contain future outbreaks.

A review of testing and assurance methods for Trichinella surveillance programs.

While global cases of trichinellosis have fallen since pork regulation began, the disease remains a danger to pork and animal game consumers as well as a liability to producers. Managing food safety risk and supporting agricultural trade requires cost-effective and sensitive diagnostic methods. Several means exist to inspect pork for parasitic infections. Here, we review literature concerning the sensitivity, specificity, and cost of these methods. We found that artificial digestion coupled with optical microscopy to be the best method for verification of Trichinella larva free pork due to its cost efficiency, high specificity, and reliability. Serological techniques such as ELISA are useful for epidemiological surveillance of swine. While current PCR techniques are quick and useful for diagnosing species-specific infections, they are not cost efficient for large-scale testing. However, as PCR techniques, including Lateral Flow- Recombinase Polymerase Amplification (LF-RPA), improve and continue to reduce cost, such methods may ultimately succeed artificial digestion.