Comprehensive Analysis of Infectious Agents Associated With Diarrhea in Foals in Central Kentucky

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1 Comprehensive Analysis of Infectious Agents Associated With Diarrhea in Foals in Central Kentucky Nathan M. Slovis, DVM, Diplomate ACVIM, CHT; Justine Elam, MT (ASCP); Marko Estrada, BS; Mary Ferrero Thao, BAS, MBA; and Christian M. Leutenegger, DVM, PhD, FVH* Enteric infectious agents are a significant cause of morbidity and mortality in the neonatal foal. Common suspects of infectious agents are traditionally tested in a stand-alone format, but using a panel of 10 polymerase chain-reaction (PCR) tests reveals coinfections to be much more common than previously reported. Well-known enteric pathogens such as Cryptosporidium and coronaviruses have to be further investigated in the foal. Authors addresses: Hagyard Medical Institute, 4250 Iron Works Pike, Lexington, Kentucky (Slovis, Elam); and IDEXX Laboratories, Inc., Molecular Diagnostics, 2825 KOVR Drive, West Sacramento, California (Estrada, Thao, Leutenegger); Christian-Leutenegger@IDEXX.com. *Presenting and corresponding author AAEP. 1. Introduction Diarrhea caused by infectious agents is common in foals, but there is no comprehensive understanding of the relative prevalence of common agents and the mechanism of coinfections. 1 Furthermore, non equine-specific test reagents used in diagnostic laboratories to detect equine viruses, protozoa, and bacteria cause true prevalence to be underestimated. In this study, we used highly sensitive and equine-specific real-time polymerase chain-reaction (PCR) tests to target nine infectious agents or their toxin genes that are able to induce or at least contribute to foal diarrhea. Included were real-time PCR assays for equine rotavirus (ERV), equine coronavirus (ECoV), Clostridium difficile toxin A (CDTA), toxin B (CDTB), Neorickettsia risticii [Potomac Horse Fever (PHF)], Clostridium perfringens enterotoxin (CPE), Lawsonia intracellularis, Rhodococcus equi, Cryptosporidium, and (PCR on selenite enrichment broth and culture). These tests were used to determine the prevalence of monoinfections and coinfections in healthy and sick foals with clinical signs of diarrhea. The objective of this study was to determine the prevalence of the nine infectious agents in sick and healthy foal populations with ages ranging from 2 to 20 wk and the degree that coinfections are associated with clinical signs of gastrointestinal (GI) disease. 2. Materials and Methods Fecal samples (approximately 5 g) were collected from GI-diseased (n 52) and healthy foals (n 37). NOTES Vol. 56 AAEP PROCEEDINGS

2 GI disease was defined as watery diarrhea and/or ultrasonic evidence of enterocolitis. Fecal material was stored and transported into fecal containers, kept at 4 C, and sent overnight to a diagnostic reference laboratory. a Fecal samples were processed immediately. Total nucleic acid extractions and real-time PCR were performed at a commercial laboratory under test code 2911 (equine diarrhea panel comprehensive). Total nucleic acid extraction protocols were used according to the manufacturer s recommendation. 2 Total nucleic acid was used to reverse-transcribe the RNA portion into complementary DNA for the RNA applications (rotavirus and coronavirus). 3 Genomic DNA (gdna) and cdna were used for real-time PCR according to published protocols. All real-time PCR assays were validated to use the same PCR reaction conditions and reagent concentrations to allow analysis of all PCR targets and quality controls on the same 384- well plate. 4 Amplification was carried out on a Roche Light Cycler (LC) 480 instrument b using the default amplification protocol: 2 min at 50 C, 10 min at 95 C, and then 45 cycles of 10 s at 95 C, 20 s at 60 C, and1sat72 C. Crossing points (CP) were calculated using the second derivative maximummethod analysis module with the high-sensitivity algorithm. Real-time PCR tests included PCR primers and a 6-FAM TAMRA quenched conventional hydrolysis probe. c Outside primers allowing confirmation of positive PCR results by sequencing of the target region were designed for all PCR tests. Target genes for the real-time PCR tests were the following: ERV, VP4 (GenBank accession number EU717544) and VP6 (L49043); ECoV, M gene (EF446615); CDTA, toxin A (X60984); CDTB, toxin B (X60984); PHF, 16s rrna (AF ) 5 ; C. perfringens, enterotoxin (AM888388); Lawsonia intracellularis, aspartate ammonia-lyase (aspa; AM180252); R. equi, virulence-associated protein A gene (vapa; AF116907) 6 ; and, invasion A gene (inva; EU348366). Commercially available PCR reagents were used for the PCR amplification. d Seven quality controls were run with each diagnostic sample, including (1) PCR positive controls, (2) PCR negative controls, (3) negative extraction controls, (4) DNA preanalytical quality control targeting the host ssr rrna (18S rrna) gene complex, (5) RNA pre-analytical quality control targeting the host ssr rrna gene complex, (6) an internal positive control spiked into the lysis solution, and (7) an environmental contamination monitoring control. These controls assessed the reliability of the PCR protocols (1 and 6), absence of contamination in the reagents (2) and laboratory (7), absences of cross-contamination during the extraction process (3), quality and integrity of the DNA and RNA as a measure of sample quality (4 and 5), reverse transcription (RT) protocol (5), and absence of PCR inhibitory substances as a carryover from the fecal matrix (6). For the culture, approximately 1gof feces was inoculated into 8 ml of selenite broth and incubated aerobically at 35 C for h. After incubation, they were subcultured onto a Hektoen plate, and suspicious black or clear colonies were streaked to Triple Sugar Iron Agar (TSI) and Lysine Iron Agar (LIA). If the biochemical tests were suspicious for, they were identified by an automated microbiology culture instrument. e Statistical analysis, contingency tables, and p values (Fisher s exact test) were determined using a commercially available statistics program. f 3. Results The overall prevalence for the presence of infectious agents was significantly associated with the GI-diseased animal group: in the GI-diseased foals, a total of 70 positive results were recorded (1.34 infectious events per animal), whereas the healthy animals tested positive in 20 tests (r 0.54 infections per animal; p ). The most frequent infectious agents isolated from healthy foals included ECoV (n 10), R. equi (n 5), Cryptosporidium (n 4), and ERV (n 1). In the GI-diseased group, the most frequent infectious agent was ERV (n 18) followed by ECoV (n 15) and Cryptosporidium (n 14). Two infectious agents were found to be significantly associated with the foals in the GI diseases group (Table 1): ERV was found with the highest prevalence in the sick foal group (35% vs. 3%; odds ratio 19.1; p ; Table 1) followed by ECoV (29% vs. 27%; odds ratio 25.8; p ). Cryptosporidium (27% vs. 11%), (13% vs. 0% by PCR and 8% vs. 0% by culture), CDTA, CDTB, PHF, and CPE seemed to be associated with sick foals as well but because of small numbers, were not significant (p 0.05). Lawsonia was not expected to be present in the population under study. R. equi shedding (with virulence gene vapa) was found in almost identical numbers in both groups. Comparing coinfections between the two groups, it is evident that the healthy group had significantly fewer coinfections (11%) than monoinfections (37%; p 0.04) (Table 2), whereas this situation was reversed in the sick group: 29% monoinfections vs. 42% coinfections (total of 71%). The ratio of monoinfections versus coinfections in the healthy group was 3:1, whereas in the GI-diseased group, the ratio was 1:1.5. Sick foals had significantly more coinfections than healthy foals (42% vs. 11%; p ). Coinfections in the sick foal group included 10 animals with two infectious agents, 8 animals with three infectious agents, and 1 animal with four infectious agents. Of the 18 positive ERV PCR tests in the sick animal group, 12 were coinfections (Table 3). Surprisingly, the combination with Cryptosporidium was the most frequent coinfection, followed by bacterial coinfections. Only one animal was infected with ERV in the healthy group. Ten ECoV infections were recorded in the healthy group, of which eight were monoinfections and only AAEP PROCEEDINGS Vol

3 Table 1. Prevalence of Infectious Agents in Sick and Healthy Foals Real-Time PCR Tests Rotavirus Coronavirus Toxin A Toxin B Neorickettsia risticii C. perfringens enterotoxin Healthy Foals 3% 27% 0% 0% 0% 0% Sick Foals 35% 29% 6% 6% 4% 8% Odds Ratio P Value Lawsonia intracellulare Rhodococcus equi Cryptosporidium Culture Healthy Foals 0% 14% 11% 0% 0% Sick Foals 0% 8% 27% 13% 8% Odds Ratio P Value All infectious agents were detected with equine specific real-time PCR tests. was also identified by culture. Odds ratios were calculated based on contingency tables, and exact p values were calculated using Fisher s exact test. two were coinfections with either R. equi or Cryptosporidium, respectively. In contrast, all 15 recorded ECoV infections in the GI-diseased group were associated with coinfections. ECoV coinfections were most frequently associated with Cryptosporidium (n 8) and/or ERV (n 7). Six of the ECoV infections were triple infections: four in combination with ERV and Cryptosporidium, one with CPE and Cryptosporidium, and one with CDTA/B and PHF. Four Cryptosporidium -positive foals were recorded in the healthy group (11%), of which three were coinfections: one with ECoV and two with R. equi. In the sick animal group, 14 positive signals were recorded (27%), of which 10 were coinfections with one or two additional agents. Of the 10 coinfections, 8 were with ECoV, and 7 were with ERV. Seven foals were triple-infected with the following combinations: four with ERV and ECoV, one with ERV and R. equi, one with ECoV and CPE, and one with ECoV and. was only detected in the sick foal group; four infected foals were detected by culture, and all four were also positive by PCR. Real-time PCR detected in three additional foals. Two of the discrepant positive signals were sequenced with outside primers and confirmed to be positive for typhimurium. The third case did not yield a strong enough positive signal to be sequenced. Four of seven -positive horses were coinfected (57%) with ERV, ECoV, CPE, or Cryptosporidium One of the positive foals was triple-infected with ECoV and Cryptosporidium, and a second foal was tripleinfected with Cryptosporidium and ECoV. 4. Discussion Many infectious diseases consist of multifactorial pathologies. Paradigmatic examples come from the immune responses elicited to the pathogens in which effects of coinfections combine together, yielding a complex mutual interaction that boosts infection progression to lethality. A well studied example is human immunodeficiency virus (HIV), which suppresses the immune system, favoring the insurgence of opportunistic infections; other examples are tuberculosis, 7 toxoplasmosis, 8 histoplasmosis, 8 hepatitis C virus, 9 and Cryptosporidium 10 infections. In veterinary medicine, the description of coinfections with GI disease has not been welldocumented. Although the understanding of the presence of coinfections exists in the veterinary community, no comprehensive knowledge of the extent and possible combinations of infectious agents and their pathogenicities in the equine GI tract exists The current study addresses the aspect of prevalence and possible combinations of infectious agents with a comprehensive panel of molecular test in combination with culture in the 2009 foaling season in Central Kentucky. The results show that (1) the rate of coinfections as a cause for diarrhea in foals has been underestimated, (2) coinfections between viruses and protozoa are almost as Table 2. Analysis of Monoinfections Versus Coinfections Compared Between Healthy and Sick Foal Groups Monoinfection Coinfection Ratio M vs. C Total Infections Infection per Animal Healthy Foals 37% 11% 3: Sick Foals 29% 42% 1: Also calculated is the ratio of monoinfections versus coinfections. animal was calculated. M, monoinfection; C, coinfection. Using the number of total recorded infections, the infection per Vol. 56 AAEP PROCEEDINGS

4 Table 3. Counts of Total Number of Infections and Number of Monoinfections and Coinfections for Each Infectious Agent by Group Type of Infection Recorded Real-Time PCR Culture Rotavirus Coronavirus Toxin A Toxin B Neorickettsia risticii C. perfringens enterotoxin Healthy Group Monoinfection Coinfection calls Sick Group Monoinfection Coinfection calls Lawsonia intracellulare Rhodococcus equi Cryptosporidium Healthy Group Monoinfection Coinfection calls Sick Group Monoinfection Coinfection calls Infectious agents were detected by either real-time PCR (all) or culture ( only). is not considered a coinfection. toxin A and B presence frequent between viruses and bacteria, (3) infectious agents not considered to be of importance or not tested for in the past were present at high prevalence, and (4) real-time PCR in combination with culture (for some bacterial targets) allows rapid, reliable, and highly specific diagnosis of infectious agents contributing to diarrhea in foals. References and Footnotes 1. Wohlfender FD, Barrelet FE, Doherr MG, et al. Diseases in neonatal foals. Part 2: potential risk factors for a higher incidence of infectious diseases during the first 30 days post partum. Vet J 2009;41: Mapes S, Leutenegger CM, Pusterla N. Nucleic acid extraction methods for detection of EHV-1 from blood and nasopharyngeal secretions. Vet Rec 2008;162: Miller WA, Gardner IA, Atwill ER, et al. Evaluation of methods for improved detection of Cryptosporidium in mussels (Mytilus californianus). J Microbiol Methods 2006; 65: Livak K, Marmaro J, Flood S. Guidelines for designing Taq- Man fluorogenic probes for 5 nuclease assays. PE Applied Biosystems Research News Pusterla N, Leutenegger CM, Sigrist B, et al. Detection and quantitation of Ehrlichia risticii genomic DNA by real-time PCR in infected horses and snails. Vet Parasitol 2000;90: Pusterla N, Wilson WD, Mapes S, et al. Diagnostic evaluation of real-time PCR in the detection of Rhodococcus equi in faeces and nasopharyngeal swabs from foals with pneumonia. Vet Rec 2007;25: Ranjbar S, Boshoff HI, Mulder A, et al. HIV-1 replication is differentially regulated by distinct clinical strains of Mycobacterium tuberculosis. PLoS 2009;1: Murata M, Furusyo N, Otaguro S, et al. HIV infection with concomitant cerebral toxoplasmosis and disseminated histoplasmosis in a 45-year-old man. J Infect Chemother 2007; 13: d Arminio Monforte A, Cozzi-Lepri A, Castagna A, et al. Risk of developing specific AIDS-defining illnesses in patients coinfected with HIV and hepatitis C virus with or without liver cirrhosis. Clin Infect Dis 2009;15: Dupont C, Bougnoux ME, Turner L, et al. Microbiological findings about pulmonary cryptosporidiosis in two AIDS patients. J Clin Microbiol 1996;34: Gal A, Harrus S, Arcoh I, et al. Coinfection with multiple tick-borne and intestinal parasites in a 6-week-old dog. Can Vet J 2007;48: Yabsley MJ, McKibben J, Macpherson CN, et al. Prevalence of Ehrlichia canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, Bartonella vinsonii berkhoffii, and Rickettsia in dogs from Grenada. Vet Parasitol 2008;14: Sasanelli M, Paradies P, Lubas G, et al. Atypical clinical presentation of coinfection with Ehrlichia, Babesia and Hepatozoon species in a dog. Vet Rec 2009;3: AAEP PROCEEDINGS Vol

5 14. Jittapalapong S, Sittisan P, Sakpuaram T, et al. Coinfection of Leptospira spp and Toxoplasma gondii among stray dogs in Bangkok, Thailand. Southeast Asian J Trop Med Public Health 2009;40: a IDEXX Laboratories, Inc., Molecular Diagnostics, West Sacramento, CA b Roche Applied Science, Indianapolis, IN c TaqMan, Eurofins MWG Operon, Huntsville, AL d Roche LightCycler R 480 Probes Master, Roche Applied Science, Indianapolis, IN e Vitek, biomerieux Inc., Durham, NC f GraphPad Prism, San Diego, CA Vol. 56 AAEP PROCEEDINGS