Genetic Tracking of a Rabid Coyote (Canis latrans) Detected beyond a Rabies Enzootic Area in West Virginia, USA.

Wildlife translocation and cross-species transmission can impede control and elimination of emerging zoonotic diseases. Tracking the geographic origin of both host and virus (i.e., translocation versus local infection) may help determine the most effective response when high-risk cases of emerging pathogens are identified in wildlife. In May 2022, a coyote (Canis latrans) infected with the raccoon (Procyon lotor) rabies virus variant (RRV) was collected in Lewis County, West Virginia, USA, an area free from RRV. We applied host population genomics and RRV phylogenetic analyses to determine the most likely geographic origin of the rabid coyote. Coyote genomic analyses included animals from multiple eastern states bordering West Virginia, with the probable origin of the rabid coyote being the county of collection. The RRV phylogenetic analyses included cases detected from West Virginia and neighboring states, with most similar RRV sequences collected in a county 80 km to the northeast, within the oral rabies vaccination zone. The combined results suggest that the coyote was infected in an RRV management area and carried the RRV to Lewis County, a pattern consistent with coyote local movement ecology. Distant cross-species transmission and subsequent host movement presents a low risk for onward transmission in raccoon populations. This information helped with emergency response decision-making, thereby saving time and resources.

Detection of Apparent Early Rabies Infection by LN34 Pan-Lyssavirus Real-Time RT-PCR Assay in Pennsylvania.

The Pennsylvania Department of Health Bureau of Laboratories (PABOL) tested 6855 animal samples for rabies using both the direct fluorescent antibody test (DFA) and LN34 pan-lyssavirus reverse transcriptase quantitative PCR (RT-qPCR) during 2017-2019. Only two samples (0.03%) were initially DFA negative but positive by LN34 RT-qPCR. Both cases were confirmed positive upon re-testing at PABOL and confirmatory testing at the Centers for Disease Control and Prevention by LN34 RT-qPCR and DFA. Rabies virus sequences from one sample were distinct from all positive samples processed at PABOL within two weeks, ruling out cross-contamination. Levels of rabies virus antigen and RNA were low in all brain structures tested, but were higher in brain stem and rostral spinal cord than in cerebellum, hippocampus or cortex. Taken together, the low level of rabies virus combined with higher abundance in more caudal brain structures suggest early infection. These cases highlight the increased sensitivity and ease of interpretation of LN34 RT-qPCR for low positive cases.

Integrated Surveillance for Antimicrobial Resistance in Salmonella From Clinical and Retail Meat Sources Reveals Genetically Related Isolates Harboring Quinolone- and Ceftriaxone-Resistant Determinants.

BACKGROUND: Antimicrobial resistance in foodborne pathogens, including nontyphoidal Salmonella (NTS), is a public health concern. Pennsylvania conducts integrated surveillance for antimicrobial resistance in NTS from human and animal sources. METHODS: During 2015-2017, clinical laboratories submitted 4478 NTS isolates from humans and 96 isolates were found in 2520 retail meat samples. One hundred nine clinical isolates that shared pulsed-field gel electrophoresis patterns with meat isolates and all strains from meat samples were tested for susceptibility to antimicrobial agents. Six clinical and 96 NTS isolates from meat sources (total 102) were analyzed by whole-genome sequencing (WGS). RESULTS: Twenty-eight (25.7%) of the 109 clinical NTS and 21 (21.9%) of strains from meat sources had resistance to ≥3 antimicrobial drug classes (multidrug resistance). Sixteen of the 102 (15.7%) isolates analyzed by WGS had resistance mechanisms that confer resistance to expanded-spectrum cephalosporins, such as ceftriaxone. We identified bla CTX-M-65 in 2 S. Infantis isolates from clinical and 3 S. Infantis isolates from meat sources. These 5 bla CTX-M-65-positive S. Infantis strains carried ≥5 additional resistance genes plus a D87Y mutation in gyrA that encodes fluoroquinolone resistance. WGS showed that isolates from patients and meat samples were within ≤10 and ≤5 alleles for S. Infantis and S. Reading, respectively. CONCLUSIONS: A significant proportion of NTS isolates from human and animal sources were multidrug resistant and 16% had genetic mechanisms that confer resistant to ceftriaxone. These results emphasize need for integrated surveillance in healthcare and agricultural settings.

Yersinia enterocolitica Outbreak Associated with Pasteurized Milk.

In July 2019, we investigated a cluster of Yersinia enterocolitica cases affecting a youth summer camp and nearby community in northeastern Pennsylvania. After initial telephone interviews with camp owners and community members, we identified pasteurized milk from a small dairy conducting on-site pasteurization, Dairy A, as a shared exposure. We conducted site visits at the camp and Dairy A where we collected milk and other samples. Samples were cultured for Y. enterocolitica. Clinical and nonclinical isolates were compared using molecular subtyping. We performed case finding, conducted telephone interviews for community cases, and conducted a cohort study among adult camp staff by administering an online questionnaire. In total, we identified 109 Y. enterocolitica cases. Consumption of Dairy A milk was known for 37 (34%); of these, Dairy A milk was consumed by 31 (84%). Dairy A had shipped 214 gallons of pasteurized milk in 5 weekly shipments to the camp by mid-July. Dairy A milk was the only shared exposure identified between the camp and community. Y. enterocolitica was isolated from Dairy A unpasteurized milk samples. Five clinical isolates from camp members, two clinical isolates from community members, and nine isolates from unpasteurized milk were indistinguishable by whole-genome sequencing. The risk for yersinosis among camp staff who drank Dairy A milk was 5.3 times the risk for those who did not (95% confidence interval: 1.6-17.3). Because Dairy A only sold pasteurized milk, pasteurized milk was considered the outbreak source. We recommend governmental agencies and small dairies conducting on-site pasteurization collaborate to develop outbreak prevention strategies.

Outbreaks of Adenovirus-associated Respiratory Illness on 5 College Campuses in the United States, 2018-2019.

BACKGROUND: Human adenoviruses (HAdVs) are commonly associated with acute respiratory illness. HAdV outbreaks are well documented in congregate military training settings, but less is known about outbreaks on college campuses. During fall 2018 and spring 2019, 5 United States (US) colleges reported increases in HAdV-associated respiratory illness. Investigations were performed to better understand HAdV epidemiology in this setting. METHODS: A case was defined as a student at one of the 5 colleges, with acute respiratory illness and laboratory-confirmed HAdV infection during October 2018-December 2018 or March-May 2019. Available respiratory specimens were typed by HAdV type-specific real-time polymerase chain reaction assays, and for a subset, whole genome sequencing was performed. We reviewed available medical records and cases were invited to complete a questionnaire, which included questions on symptom presentation, social history, and absenteeism. RESULTS: We identified 168 HAdV cases. Median age was 19 (range, 17-22) years and 102 cases (61%) were male. Eleven cases were hospitalized, 10 with pneumonia; 2 cases died. Among questionnaire respondents, 80% (75/94) missed ≥ 1 day of class because of their illness. Among those with a type identified (79%), HAdV types 4 and 7 were equally detected, with frequency of each varying by site. Genome types 4a1 and 7d were identified, respectively, by whole genome sequence analysis. CONCLUSIONS: HAdV respiratory illness was associated with substantial morbidity and missed class time among young, generally healthy adults on 5 US college campuses. HAdVs should be considered a cause of respiratory illness outbreaks in congregate settings such as college campuses.

Detection and Characterization of Salmonella spp. in Raw Commingled Bulk Tank Milk from Dairies in Pennsylvania.

A microbiological survey was conducted to determine the presence of Salmonella spp. in raw commingled bulk tank milk (BTM) collected from the Pennsylvania dairies intended for pasteurization. The survey found 8.1% (10/123) of samples positive for Salmonella. Salmonella Cerro was the predominant serovar and genetic analysis of the Salmonella Cerro showed the existence of diverse yet closely related genotypes. Antibiotic susceptibility testing conducted on all isolates showed pan-susceptible pattern against 15 drugs covering 9 drug classes. The study helped determine the presence of Salmonella spp. in the commingled BTM, antibiotic susceptibility patterns, and serovars along with genotypic diversity among the predominant serovar, Salmonella Cerro.

Adenovirus-Associated Influenza-Like Illness among College Students, Pennsylvania, USA.

Among students with influenza-like illness at a Pennsylvania college student health center during 2016-2017, 44 (15%) of 288 with respiratory specimens tested positive for human adenovirus (HAdV). HAdV-3, -7, and -4 predominated, and types clustered temporally. HAdV infection should be considered among college students with acute respiratory illness.

Multi-site evaluation of the LN34 pan-lyssavirus real-time RT-PCR assay for post-mortem rabies diagnostics.

Rabies is a fatal zoonotic disease that requires fast, accurate diagnosis to prevent disease in an exposed individual. The current gold standard for post-mortem diagnosis of human and animal rabies is the direct fluorescent antibody (DFA) test. While the DFA test has proven sensitive and reliable, it requires high quality antibody conjugates, a skilled technician, a fluorescence microscope and diagnostic specimen of sufficient quality. The LN34 pan-lyssavirus real-time RT-PCR assay represents a strong candidate for rabies post-mortem diagnostics due to its ability to detect RNA across the diverse Lyssavirus genus, its high sensitivity, its potential for use with deteriorated tissues, and its simple, easy to implement design. Here, we present data from a multi-site evaluation of the LN34 assay in 14 laboratories. A total of 2,978 samples (1,049 DFA positive) from Africa, the Americas, Asia, Europe, and the Middle East were tested. The LN34 assay exhibited low variability in repeatability and reproducibility studies and was capable of detecting viral RNA in fresh, frozen, archived, deteriorated and formalin-fixed brain tissue. The LN34 assay displayed high diagnostic specificity (99.68%) and sensitivity (99.90%) when compared to the DFA test, and no DFA positive samples were negative by the LN34 assay. The LN34 assay produced definitive findings for 80 samples that were inconclusive or untestable by DFA; 29 were positive. Five samples were inconclusive by the LN34 assay, and only one sample was inconclusive by both tests. Furthermore, use of the LN34 assay led to the identification of one false negative and 11 false positive DFA results. Together, these results demonstrate the reliability and robustness of the LN34 assay and support a role for the LN34 assay in improving rabies diagnostics and surveillance.

CRISPR Typing and Antibiotic Resistance Correlates with Polyphyletic Distribution in Human Isolates of Salmonella Kentucky.

Although infrequently associated with reported salmonellosis in humans, Salmonella enterica, subsp. enterica serovar Kentucky (ser. Kentucky) is the most common nonclinical, nonhuman serovar reported in the United States. The goal of this study was to use Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-multi-virulence-locus sequence typing (MVLST) to subtype a collection of human clinical isolates of ser. Kentucky submitted to the Pennsylvania Department of Health and to determine the extent of antibiotic resistance in these strains. This analysis highlighted the polyphyletic nature of ser. Kentucky, and separated our isolates into two groups, Group I and Group II, which were equally represented in our collection. Furthermore, antimicrobial susceptibility testing on all isolates using a National Antimicrobial Resistance Monitoring System (NARMS) panel of antibiotics demonstrated that resistance profiles could be divided into two groups. Group I isolates were resistant to cephems and penicillins, whereas Group II isolates were resistant to quinolones, gentamicin, and sulfisoxazole. Collectively, 50% of isolates were resistant to three or more classes of antibiotics and 30% were resistant to five or more classes. The correlation of antibiotic resistance with the two different lineages may reflect adaptation within two distinct reservoirs of ser. Kentucky, with differential exposure to antimicrobials.

The combination of CRISPR-MVLST and PFGE provides increased discriminatory power for differentiating human clinical isolates of Salmonella enterica subsp. enterica serovar Enteritidis.

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is a major cause of foodborne salmonellosis. Rapid, efficient and accurate methods for identification are required to track specific strains of S. Enteritidis during outbreaks of human salmonellosis. By exploiting the hypervariable nature of virulence genes and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs), we previously developed a powerful sequence-based subtyping approach, designated CRISPR-MVLST. To substantiate the applicability of CRISPR-MVLST, we analyzed a broad set of S. Enteritidis isolates collected over a six-year period. Among 141 isolates we defined 22 Enteritidis Sequence Types (ESTs), the majority of which were novel. Notably, strains exhibiting the common PFGE pattern, JEGX01.0004 (characteristic of ∼40% of S. Enteritidis isolates in the United States), were separated into twelve distinct sequence types. Conversely, isolates of EST4, the most predominant EST we observed, comprised eight different PFGE patterns. Importantly, we showed that some genotypes that were previously associated with the food supply chain at the farm level have now been identified in clinical samples. CRISPR sequence data shows subtle but distinct differences among different alleles of S. Enteritidis, suggesting that evolution of these loci occurs vertically, as opposed to previously reported evolution by spacer acquisition in other bacteria.

Culturing stool specimens for Campylobacter spp., Pennsylvania, USA.

In 2010, we surveyed 176 clinical laboratories in Pennsylvania regarding stool specimen testing practices for enteropathogens, including Campylobacter spp. Most (96.3%) routinely test for Campylobacter spp. In 17 (15.7%), a stool antigen test is the sole method for diagnosis. We recommend that laboratory practice guidelines for Campylobacter spp. testing be developed.