Dietary monoglyceride supplementation to support intestinal integrity and host defenses in health-challenged weanling pigs.

Frequent incidence of postweaning enterotoxigenic Escherichia coli (ETEC) diarrhea in the swine industry contributes to high mortality rates and associated economic losses. In this study, a combination of butyric, caprylic, and capric fatty acid monoglycerides was investigated to promote intestinal integrity and host defenses in weanling pigs infected with ETEC. A total of 160 pigs were allotted to treatment groups based on weight and sex. Throughout the 17-d study, three treatment groups were maintained: sham-inoculated pigs fed a control diet (uninfected control [UC], n = 40), ETEC-inoculated pigs fed the same control diet (infected control [IC], n = 60), and ETEC-inoculated pigs fed the control diet supplemented with monoglycerides included at 0.3% of the diet (infected supplemented [MG], n = 60). After a 7-d acclimation period, pigs were orally inoculated on each of three consecutive days with either 3 mL of a sham-control (saline) or live ETEC culture (3 × 109 colony-forming units/mL). The first day of inoculations was designated as 0 d postinoculation (DPI), and all study outcomes reference this time point. Fecal, tissue, and blood samples were collected from 48 individual pigs (UC, n = 12; IC, n = 18; MG, n = 18) on 5 and 10 DPI for analysis of dry matter (DM), bacterial enumeration, inflammatory markers, and intestinal permeability. ETEC-inoculated pigs in both the IC and MG groups exhibited clear signs of infection including lower (P < 0.05) gain:feed and fecal DM, indicative of excess water in the feces, and elevated (P < 0.05) rectal temperatures, total bacteria, total E. coli, and total F18 ETEC during the peak-infection period (5 DPI). Reduced (P < 0.05) expression of the occludin, tumor necrosis factor α, and vascular endothelial growth factor A genes was observed in both ETEC-inoculated groups at the 5 DPI time point. There were no meaningful differences between treatments for any of the outcomes measured at 10 DPI. Overall, all significant changes were the result of the ETEC infection, not monoglyceride supplementation.

Infection caused by the bacterium known as enterotoxigenic Escherichia coli (ETEC) is a common disruptor of weaned pigs' health, leading to economic losses for the producers. To determine if nutritional supplementation could help protect against these losses, weaned pigs were assigned to one of three treatments: 1) uninfected and fed a standard nursery pig diet, 2) infected with ETEC and fed the same standard diet, or 3) infected with ETEC and fed the standard diet supplemented with a combination of butyric, caprylic, and capric fatty acid monoglycerides. Growth performance was tracked throughout the 17-d study and health outcomes were measured at the peak and resolution of ETEC infection. At the peak-infection time point, pigs that were infected with ETEC had lower fecal moisture content, greater fecal bacterial concentrations, and elevated body temperatures compared with uninfected pigs. Additionally, infection reduced expression of genes related to inflammation, angiogenesis, and the intestinal barrier during the peak-infection period. Overall, all significant changes were the result of the ETEC infection, and there were no meaningful differences observed between the different treatments.

Development of a new loop-mediated isothermal amplification test for the sensitive, rapid, and economic detection of different genotypes of Classical swine fever virus.

Background: Classical swine fever virus (CSFV) remains one of the most important pathogens in animal health. Pathogen detection relies on viral RNA extraction followed by RT-qPCR. Novel technologies are required to improve diagnosis at the point of care. Methods: A loop-mediated isothermal amplification (LAMP) PCR technique was developed, with primers designed considering all reported CSFV genotypes. The reaction was tested using both fluorometric and colorimetric detection, in comparison to the gold standard technique. Viral strains from three circulating CSFV genotypes were tested, as well as samples from infected animals. Other pathogens were also tested, to determine the LAMP specificity. Besides laboratory RNA extraction methods, a heating method for RNA release, readily available for adaptation to field conditions was evaluated. Results: Three primer sets were generated, with one of them showing better performance. This primer set proved capable of maintaining optimal performance at a wide range of amplification temperatures (60°C - 68°C). It was also able to detect CSFV RNA from the three genotypes tested. The assay was highly efficient in detection of samples from animals infected with field strains from two different genotypes, with multiple matrices being detected using both colorimetric and fluorometric methods. The LAMP assay was negative for all the unrelated pathogens tested, including Pestiviruses. The only doubtful result in both fluorometric and colorimetric LAMP was against the novel Pestivirus italiaense, ovine Italy Pestivirus (OVPV), which has proven to have cross-reaction with multiple CSFV diagnostic techniques. However, it is only possible to detect the OVPV in a doubtful result if the viral load is higher than 10000 viral particles. Conclusion: The results from the present study show that LAMP could be an important addition to the currently used molecular diagnostic techniques for CSFV. This technique could be used in remote locations, given that it can be adapted for successful use with minimal equipment and minimally invasive samples. The joined use of novel and traditional diagnostic techniques could prove to be a useful alternative to support the CSF control.

Human exposures to Brucella canis from a pregnant dog during an international flight: Public health risks, diagnostic challenges and future considerations.

AIMS: This report documents the exposure of passengers and crew of a commercial international flight to the zoonotic pathogen Brucella canis after an infected dog aborted in the passenger cabin of the aircraft. This case demonstrates the challenges associated with brucellosis screening and the risks that airline personnel, airport employees and travellers face when animals with unrecognized zoonotic infections are transported. METHODS/RESULTS: The public health investigation of this case was conducted by the Centers for Disease Control, the Illinois Department of Health and the Illinois Department of Agriculture, in collaboration with a local veterinary clinic and several academic and federal diagnostic laboratories. It included an extensive diagnostic evaluation of the dam and aborted foetuses to confirm a diagnosis of canine brucellosis. Passengers, airline personnel and staff from the veterinary clinic where the dogs were treated underwent risk assessments, and clinic staff also received detailed guidance regarding infection prevention practices. CONCLUSIONS: Animal shelters and breeding programs are recommended to screen dogs routinely for brucellosis, but it is not unusual for domestic or imported animals to have unknown health histories, including the dog's brucellosis status, at the time of purchase, adoption, or re-homing. Testing recommendations and requirements vary by state, making it challenging for state public health and animal health agencies to monitor and respond appropriately. This case highlights the importance of Brucella spp. screening in sexually intact dogs prior to breeding, purchase, or domestic or international transportation of the dogs. The transportation of pregnant dogs may present a previously unrecognized public health threat in addition to contributing to unnecessary stress and health risks for pregnant animals.

Assessing the urinary concentration of nitrofurantoin and its antibacterial activity against Escherichia coli, Staphylococcus pseudintermedius, and Enterococcus faecium isolated from dogs with urinary tract infections.

Nitrofurantoin, a broad-spectrum nitrofuran class antibiotic, is applied as a first-line antibiotic in treating human urinary tract infections (UTIs) due to its great efficacy and high achievable concentration. The interest in using this antibiotic in companion animals has increased due to the growing demand for effective antibiotics to treat UTIs caused by multidrug-resistant bacteria. Currently, the susceptibility interpretations for nitrofurantoin are based on the breakpoints set for humans, while the canine-specific breakpoints are still unavailable. In this study, we assessed the concentration of nitrofurantoin reaching the dog's urine using the recommended oral dosing regimen. In addition, we examined the efficacy of this breakpoint concentration against the common canine UTI pathogens, Escherichia coli, Staphylococcus pseudintermedius, and Enterococcus faecium. Eight experimental beagle dogs were treated with ~5 mg/kg of nitrofurantoin macrocrystal PO 8qh for 7 days. The urine samples were collected via cystocentesis at 2, 4, and 6 h after administration on day 2 and day 7 and used to quantify nitrofurantoin concentrations by ultra-high performance liquid chromatography. The results showed that 26.13-315.87 µg/mL nitrofurantoin was detected in the dogs' urine with a mean and median concentration of 104.82 and 92.75 µg/mL, respectively. Additionally, individual dogs presented with urinary nitrofurantoin concentrations greater than 64 µg/mL for at least 50% of the dosing intervals. This concentration efficiently killed E. coli, and S. pseudintermedius, but not E. faecium strains carrying an MIC90 value equal to 16, 16, and 128 µg/mL, respectively. Taken together, these results suggest that the value of 64 µg/mL may be set as a breakpoint against UTI pathogens, and nitrofurantoin could be an effective therapeutic drug against E. coli and S. pseudintermedius for canine UTIs.

Efficient detection of African Swine Fever Virus using minimal equipment through a LAMP PCR method.

African swine fever virus (ASFV) currently represents the biggest threat to the porcine industry worldwide, with high economic impact and severe animal health and welfare concerns. Outbreaks have occurred in Europe and Asia since ASFV was reintroduced into the continent in 2007 and, in 2021, ASFV was detected in the Caribbean, raising alarm about the reemergence of the virus in the Americas. Given the lack of vaccines against ASFV, control of the virus relies on molecular surveillance, which can be delayed due to the need for sample shipment to specialized laboratories. Isothermal PCR techniques, such as LAMP, have become increasingly attractive as point-of-care diagnostic tools given the minimal material expense, equipment, and training required. The present study aimed to develop a LAMP assay for the detection of ASFV. Four LAMP primer sets were designed, based on a consensus sequence for the ASFV p72 gene, and were tested using a synthetic plasmid containing the cloned ASFV p72 target gene as a positive control. Two primer sets, were selected for further validation, given their very short time for amplification. Both primer sets showed thermal stability, amplifying the ASFV DNA at temperatures between 60-70°C and proved to have an analytical limit of detection as low as one ASFV-plasmid DNA copy/µL, using both fluorometric and colorimetric methods. The selected primers did not yield false positive or cross reactive results with other common swine pathogens, showing high specificity. Testing of DNA-spiked samples showed that LAMP amplification was not affected by the nature of the matrices, including oral fluids, tonsils, blood, or rectal swabs. The primer sets were able to detect the two more prevalent ASFV genotypes in the field. Taken together, the results show that ASFV-LAMP-BG2 and ASFV-LAMP-BG3 would be a useful tool for rapid, highly sensitive on-site diagnostic testing.

Canine brucellosis in three littermates, case report.

Three adult littermates were diagnosed with Brucella canis, two of which were diagnosed with discospondylitis. The first littermate, a 2-year-old spayed-female Labrador Retriever, was evaluated for progressive episodes of cervical pain, lethargy, reported circling to the right, and a right-sided head tilt. Magnetic resonance imaging (MRI) of the cervical spine revealed changes consistent with discospondylitis at C6-C7. MRI of the brain was unremarkable and cerebrospinal fluid analysis was declined. Brucella spp. was isolated from aerobic and Brucella blood cultures. PCR performed on the isolate identified Brucella canis and indirect fluorescent antibody (IFA) testing for Brucella canis also confirmed the species. Patient #1 was treated with doxycycline and marbofloxacin for 1 year. Clinical signs returned 2-years after diagnosis. Following the diagnosis of patient #1, a known littermate (patient #2) was tested for Brucella canis. Patient #2 was 2 years old and asymptomatic at the time of diagnosis. Aerobic and Brucella spp. cultures, PCR, and IFA were obtained and were diagnostic for Brucella canis. A 6-month course of marbofloxacin and doxycycline was implemented. The patient remained PCR positive following 4 months of treatment and repeat cultures were planned following 6 months of treatment; however, the patient was lost to follow-up. A third littermate (patient #3) was identified by the family of patient #1. Patient #3 was evaluated at 18 months of age for a 6-month history of progressive lumbosacral pain. Spinal radiographs revealed discospondylitis of the C3-C4, T12-T13, and L7-S1 vertebral endplates. Computed tomography (CT) of the lumbosacral spine was also consistent with discospondylitis at L7-S1. Brucella canis serologic testing consisting of rapid slide agglutination test, 2ME-rapid slide agglutination test, and cytoplasmic agar gel immunodiffusion was positive. Enrofloxacin was administered for 7 months and was discontinued thereafter based on radiographic evidence of healing and resolution of clinical signs. Although Brucella canis is not a rare disease in dogs, the documentation of two out of three adult littermates with associated discospondylitis is an interesting feature. In addition, this report highlights available diagnostic and treatment options, as each patient was managed differently based on clinical signs and the preference of the managing clinician.

Antimicrobial Resistance in Bacteria Isolated From Canine Urine Samples Submitted to a Veterinary Diagnostic Laboratory, Illinois, United States.

The emergence of antimicrobial resistance (AMR) in dogs constitutes a threat to animal and human health. There is a lack of studies in Illinois that evaluated the prevalence of AMR among urinary bacterial pathogens. In the study, we included 803 isolates (299 Gram-positive and 504 Gram-negative) that were isolated from 2,583 canine urine samples submitted to the Veterinary Diagnostic Laboratory, the University of Illinois between 2019 and 2020 from dogs suspected of urinary tract infections (UTI). The most common Gram-positive isolates included Staphylococcus pseudintermedius (17.93%), Enterococcus faecalis (9.46%), Streptococcus canis (6.10%), and Enterococcus faecium (3.74%), while Gram-negative isolates included Escherichia coli (45.58%), Proteus mirabilis (11.08%), Klebsiella pneumoniae (3.11%), and Pseudomonas aeruginosa (2.99%). Among the Gram-positive isolates, Staphylococcus pseudintermedius isolates showed a very high prevalence of resistance to penicillin (56.94%), a high prevalence of resistance to trimethoprim-sulfamethoxazole (31.94%), enrofloxacin (29.17%), and oxacillin (27.08%). Among Gram-negative bacteria, Escherichia coli isolates showed a high prevalence of resistance to ampicillin (31.42%). Considering the high prevalence of resistance to antimicrobials commonly used to treat UTI in dogs, urine samples should be collected for bacterial culture and susceptibility testing before treatment initiation to prevent treatment failures and the development of multidrug resistance. Given the possibility of zoonotic transmission of antimicrobial-resistant bacteria, veterinarians when treating UTI cases, should inform dog owners of the potential transmission risk.

Brucella ceti sequence type 23, 26, and 27 infections in North American cetaceans.

Brucella ceti infection is associated with a variety of disease outcomes in cetaceans globally. Multiple genotypes of B. ceti have been identified. This retrospective aimed to determine if specific lesions were associated with different B. ceti DNA sequence types (STs). Characterization of ST was performed on 163 samples from 88 free-ranging cetaceans, including common bottlenose dolphin Tursiops truncatus (T.t.; n = 73), common short-beaked dolphin Delphinus delphis (D.d.; n = 7), striped dolphin Stenella coeruleoalba (n = 3), Pacific white-sided dolphin Lagenorhynchus obliquidens (n = 2), sperm whale Physeter macrocephalus (n = 2), and harbour porpoise Phocoena phocoena (n = 1), that stranded along the coast of the US mainland and Hawaii. ST was determined using a previously described insertion sequence 711 quantitative PCR. Concordance with 9-locus multi-locus sequence typing was assessed in a subset of samples (n = 18). ST 26 was most commonly identified in adult dolphins along the US east coast with non-suppurative meningoencephalitis (p = 0.009). Animals infected with ST 27 were predominately perinates that were aborted or died shortly after birth with evidence of in utero pneumonia (p = 0.035). Reproductive tract inflammation and meningoencephalitis were also observed in adult T.t. and D.d. with ST 27, though low sample size limited interpretation. ST 23 infections can cause disease in cetacean families other than porpoises (Phocoenidae), including neurobrucellosis in D.d. In total, 11 animals were potentially infected with multiple STs. These data indicate differences in pathogenesis among B. ceti STs in free-ranging cetaceans, and infection with multiple STs is possible.

Effect of TrisEDTA and Chlorhexidine 0.12% on an In Vitro-Defined Biofilm Representing the Subgingival Plaque Biofilm of the Dog.

This study was designed to investigate the effects of chlorhexidine 0.12%, TrisEDTA (tromethamine ethylenediamintetraacetic acid), and a combination of chlorhexidine 0.12% and TrisEDTA on an in vitro plaque biofilm model comprised of three bacterial species commonly found in canine subgingival plaque. Porphyromonas gulae, Actinomyces canis, and Neisseria canis were grown in a biofilm on polished hydroxyapatite coated titanium alloy pucks for 72 h prior to exposure to one of four test solutions: TrisEDTA, chlorhexidine 0.12%, a combination of TrisEDTA and chlorhexidine 0.12%, or sterile deionized water as a control. Following exposure to the test solution, a sample was collected of the biofilm either immediately or following 24 h of additional incubation in a broth medium. Lower numbers of CFU/mL of Porphyromonas gulae resulted when the biofilm was treated with a solution of chlorhexidine 0.12% and TrisEDTA compared to with chlorhexidine 0.12% alone, TrisEDTA alone, or the control and so this solution can be said to be synergistic against Porphyromonas gulae in this controlled in vitro model. Greater reductions in the numbers of CFU/mL of Actinomyces canis and Neisseria canis resulted from treatment with chlorhexidine 0.12% alone than if treated with the combination of TrisEDTA and chlorhexidine 0.12%. When treated biofilm samples were allowed 24 h of additional growth in fresh media, greater variance resulted and this variance highlights the complex dynamics involved in bacterial growth within a biofilm.

PREVALENCE AND ANTIMICROBIAL RESISTANCE PATTERNS OF SALMONELLA SPP. IN TWO FREE-RANGING POPULATIONS OF EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA).

Salmonellosis is an important zoonotic infection, and exposure to pet reptiles has been implicated in several human outbreaks. Although several studies report a low prevalence of salmonellae in free-ranging chelonians, they may serve as a reservoir. In spring and summer of 2013 and 2019, free-ranging eastern box turtles (Terrapene carolina carolina) from populations in Illinois (rural) and Tennessee (urban) were collected through canine and visual search. Cloacal swab samples were collected from each turtle, selectively enriched with tetrathionate broth, then plated on selective and differential media to isolate Salmonella spp. Genus was confirmed via MALDI-TOF MS and antibiotic sensitivities were performed. Isolates were serotyped by the National Veterinary Services Laboratory. Of the 341 turtles sampled, Salmonella spp. were detected in nine individuals (2.64%; 95% CI: 1.2-5.0%). The isolates included five different serovars: Anatum (n = 2), Newport (n = 2), Thompson (n = 1), Bareilly (n = 2), and Hartford (n = 2). Salmonella spp. were detected from six animals in 2013 (3.19%, 95% CI: 1.2-6.8%) and three in 2019 (1.96%, 95% CI: 0.4-5.6%). There was no significant difference in prevalence between state, (P = 0.115), Illinois locations (P = 0.224), season (P = 0.525), year (P = 0.297), sex (P = 0.435), or age class (P = 0.549). The health of Salmonella-positive and -negative turtles was not significantly different, as assessed through hematology and plasma biochemistry (P > 0.05), indicating asymptomatic carrier status. The low prevalence detected in this study likely concludes that free-ranging eastern box turtles play a minimal role in the spread of salmonellae. However, the identified serotypes are potentially human- and animal-pathogenic. Documenting the prevalence of Salmonella serotypes in animal indicators furthers our understanding of their spread between humans, animal agriculture, and the environment.

Survey of dermatophytes in stray dogs and cats with and without skin lesions in Puerto Rico and confirmed with MALDI-TOF MS.

Dermatophytosis is a common and highly contagious zoonotic skin disease in companion animals. This disease is a major concern in geographical areas that contain large numbers of stray animal populations. Numerous surveys on dermatophytosis among stray animal populations worldwide range between 27% to 50%. In recent years, the US territory of Puerto Rico was impacted by several natural disasters such as hurricanes, which has led to a large increase of abandonment cases and an increase in the stray animal population. Due to this, large low-cost spay/neuter clinics and trap-neuter-release programs have become a more common practice on the island. During these events, veterinary staff are exposed to multiple animals with no health history, and therefore, zoonotic diseases are of concern. The aim of this study was to provide information regarding the presence of dermatophyte species in symptomatic and asymptomatic stray dogs and cats in a region of Puerto Rico. Hair samples were collected from 99 stray animals with and without dermatological clinical signs. The hair samples were cultured on plates containing rapid sporulation medium and dermatophyte test medium. All cultures were evaluated microscopically to confirm the presence of dermatophytes. Then, all dermatophytes were further evaluated with MALDI-TOF MS to compare both diagnostic tests. A total of 19 animals (19%) were positive for dermatophyte growth. Of these animals, 18/19 were infected with M. canis and 1/19 with Trichophyton spp. Animals with clinical lesions were positive only 13.5% of the time compared to asymptomatic animals, who were positive in 36% of the sample population. All 19 dermatophytes (100%) diagnosed with microscopic evaluation were confirmed with MALDI-TOF MS. Our results indicate that there is a prevalence of 19% of dermatophytosis among the stray dog and cat population of the southeastern coast of the island.

Pulmonary Mycosis Drives Forkhead Box Protein A2 Degradation and Mucus Hypersecretion through Activation of the Spleen Tyrosine Kinase-Epidermal Growth Factor Receptor-AKT/Extracellular Signal-Regulated Kinase 1/2 Signaling.

Pulmonary mycoses are difficult to treat and detrimental to patients. Fungal infections modulate the lung immune response, induce goblet cell hyperplasia and metaplasia, and mucus hypersecretion in the airways. Excessive mucus clogs small airways and reduces pulmonary function by decreasing oxygen exchange, leading to respiratory distress. The forkhead box protein A2 (FOXA2) is a transcription factor that regulates mucus homeostasis in the airways. However, little is known whether pulmonary mycosis modulates FOXA2 function. Herein, we investigated whether Blastomyces dermatitidis and Histoplasma capsulatum-infected canine and feline lungs and airway epithelial cells could serve as higher animal models to examine the relationships between fungal pneumonia and FOXA2-regulated airway mucus homeostasis. The results indicate that fungal infection down-regulated FOXA2 expression in airway epithelial cells, with concomitant overexpression of mucin 5AC (MUC5AC) and mucin 5B (MUC5B) mucins. Mechanistic studies reveal that B. dermatitidis infection, as well as ß-glucan exposure, activated the Dectin-1-SYK-epidermal growth factor receptor-AKT/extracellular signal-regulated kinase 1/2 signaling pathway that inhibits the expression of FOXA2, resulting in overexpression of MUC5AC and MUC5B in canine airway cells. Further understanding of the role of FOXA2 in mucus hypersecretion may lead to novel therapeutics against excessive mucus in both human and veterinary patients with pulmonary mycosis.

Staphylococcal Protein A (spa) Locus Is a Hot Spot for Recombination and Horizontal Gene Transfer in Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is a major canine pathogen but also occasionally colonizes and infects humans. Multidrug-resistant methicillin-resistant S. pseudintermedius (MDR MRSP) strains have emerged globally, making treatment and control of this pathogen challenging. Sequence type 71 (ST71), ST68, and ST45 are the most widespread and successful MDR MRSP clones. The potential genetic factors underlying the clonal success of these and other predominant clones remain unknown. Characterization of the pangenome, lineage-associated accessory genes, and genes acquired through horizontal gene transfer from other bacteria is important for identifying such factors. Here, we analyzed genome sequence data from 622 S. pseudintermedius isolates to investigate the evolution of pathogenicity across lineages. We show that the predominant clones carry one or more lineage-associated virulence genes. The gene encoding staphylococcal protein A (SpA), a key virulence factor involved in immune evasion and a potential vaccine antigen, is deleted in 62% of isolates. Most importantly, we have discovered that the spa locus is a hot spot for recombination and horizontal gene transfer in S. pseudintermedius, where genes related to restriction modification, prophage immunity, mercury resistance, and nucleotide and carbohydrate metabolism have been acquired in different lineages. Our study also establishes that ST45 is composed of two distinct sublineages that differ in their accessory gene content and virulence potential. Collectively, this study reports several previously undetected lineage-associated genetic factors that may have a role in the clonal success of the major MDR MRSP clones. These data provide a framework for future experimental studies on S. pseudintermedius pathogenesis and for developing novel therapeutics against this pathogen.IMPORTANCEStaphylococcus pseudintermedius is a major canine pathogen but can also occasionally infect humans. Identification of genetic factors contributing to the virulence and clonal success of multidrug-resistant S. pseudintermedius clones is critical for the development of therapeutics against this pathogen. Here, we characterized the genome sequences of a global collection of 622 S. pseudintermedius isolates. We show that all major clones, besides carrying core virulence genes, which are present in all strains, carry one or more lineage-specific genes. Many of these genes have been acquired from other bacterial species through a horizontal gene transfer mechanism. Importantly, we have discovered that the staphylococcal protein A gene (spa), a widely used marker for molecular typing of S. pseudintermedius strains and a potential vaccine candidate antigen, is deleted in 62% of strains. Furthermore, the spa locus in S. pseudintermedius acts as a reservoir to accumulate lineage-associated genes with adaptive functions.

A Validated Multiplex Real-Time PCR Assay for the Diagnosis of Infectious Leptospira spp.: A Novel Assay for the Detection and Differentiation of Strains From Both Pathogenic Groups I and II.

Leptospirosis is recognized as the most globally widespread reemerging zoonosis and represents a serious threat for both human and animal health. Indeed, leptospirosis is linked to more than 60,000 human deaths per year and to incalculable economic burden as consequence of medical treatment costs and livestock loss. The increasing number of reports from species of pathogenic Leptospira spp. group II causing disease in both humans and animals constitutes an additional concern to the complex epidemiology of this zoonotic agent. Diagnostic methods based on qPCR have improved the diagnosis of Leptospira spp. in terms of cost, time, and reliability, but most of the validated assays fail to detect species from the pathogenic group II. Hence, the current study was aimed to develop and validate a novel multiplex qPCR to enable the specific and selective detection of the whole group of infectious Leptospira spp., including both pathogenic groups I and II and moreover, selectively discriminate between them. To fit the "fitness of purpose" for the specific detection of infectious Leptospira spp. and further discrimination between both pathogenic groups three target regions on the 16S RNA gene were selected. These targets facilitated a broad and selective spectrum for the detection of all infectious Leptospira spp. with the exclusion of all saprophytic groups and the novel clade of environmental Leptospira spp. The analytical sensitivity (ASe) showed by the new assay also enables a wide window of detection for the agent at different stages of infection since the assay was able to efficiently detect at 95% of confidence ∼5 leptospires/reaction. From the evaluation of the analytical specificity (ASp) by in silico and in vitro approaches, it was congruently revealed that the primers and probes selected only recognized the specific targets for which the assay was intended. Bayesian latent class analysis of performance of the new assay on 684 clinical samples showed values of diagnostic sensitivity of 99.8% and diagnostic specificity of 100%. Thus, from the evaluation of the analytical and diagnostic parameters, the new multiplex qPCR assay is a reliable method for the diagnosis of Leptospira spp.

Prophage-Mediated Disruption of Genetic Competence in Staphylococcus pseudintermedius.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is a major cause of soft tissue infections in dogs and occasionally infects humans. Hypervirulent multidrug-resistant (MDR) MRSP clones have emerged globally. The sequence types ST71 and ST68, the major epidemic clones of Europe and North America, respectively, have spread to other regions. The genetic factors underlying the success of these clones have not been investigated thoroughly. Here, we performed a comprehensive genomic analysis of 371 S. pseudintermedius isolates to dissect the differences between major clonal lineages. We show that the prevalence of genes associated with antibiotic resistance, virulence, prophages, restriction-modification (RM), and CRISPR/Cas systems differs significantly among MRSP clones. The isolates with GyrA+GrlA mutations, conferring fluoroquinolone resistance, carry more of these genes than those without GyrA+GrlA mutations. ST71 and ST68 clones carry lineage-specific prophages with genes that are likely associated with their increased fitness and virulence. We have discovered that a prophage, SpST71A, is inserted within the comGA gene of the late competence operon comG in the ST71 lineage. A functional comG is essential for natural genetic competence, which is one of the major modes of horizontal gene transfer (HGT) in bacteria. The RM and CRISPR/Cas systems, both major genetic barriers to HGT, are also lineage specific. Clones harboring CRISPR/Cas or a prophage-disrupted comG exhibited less genetic diversity and lower rates of recombination than clones lacking these systems. After Listeria monocytogenes, this is the second example of prophage-mediated competence disruption reported in any bacteria. These findings are important for understanding the evolution and clonal expansion of MDR MRSP clones.IMPORTANCE Staphylococcus pseudintermedius is a bacterium responsible for clinically important infections in dogs and can infect humans. In this study, we performed genomic analysis of 371 S. pseudintermedius isolates to understand the evolution of antibiotic resistance and virulence in this organism. The analysis covered significant reported clones, including ST71 and ST68, the major epidemic clones of Europe and North America, respectively. We show that the prevalence of genes associated with antibiotic resistance, virulence, prophages, and horizontal gene transfer differs among clones. ST71 and ST68 carry prophages with novel virulence and antibiotic resistance genes. Importantly, site-specific integration of a prophage, SpST71A, has led to the disruption of the genetic competence operon comG in ST71 clone. A functional comG is essential for the natural uptake of foreign DNA and thus plays an important role in the evolution of bacteria. This study provides insight into the emergence and evolution of antibiotic resistance and virulence in S. pseudintermedius, which may help in efforts to combat this pathogen.

EVIDENCE OF LEPTOSPIRA SEROVARS IN WILDLIFE AND LEPTOSPIRAL DNA IN WATER SOURCES IN A NATURAL AREA IN EAST-CENTRAL ILLINOIS, USA.

We identified seven Leptospira serovars in wildlife and the presence of leptospiral DNA in water sources at a natural area within a fragmented habitat in Illinois, US. These serovars have been implicated in domestic animal and human leptospirosis, a reemerging zoonotic disease, whose reservoirs include wildlife and domestic animals. We live trapped medium-sized mammals (n=351) near building (H-sites) or forest sites (F-sites). Using serology, we evaluated exposure to Leptospira (L. interrogans serovars Autumnalis, Bratislava, Canicola, Icterohaemorrhagiae, Pomona; L. kirschneri serovar Grippotyphosa; L. borgpetersenii serovar Hardjo). Using PCR, we tested for the presence of leptospires in eight water samples (ponds, creeks, and rainwater runoff) collected near trapping sites. We identified antibody titers in raccoons (Procyon lotor; 121/221) and Virginia opossums (Didelphis virginiana; 60/112), but not in feral cats (Felis catus; 0/18). We found significant differences in overall Leptospira seroprevalence between years (P=0.043) and animal's age in 2008 (P=0.005) and 2009 (P=0.003). Serovars Autumnalis, Bratislava, and Grippotyphosa showed significant differences among age groups with the highest seroprevalence in adults. Females had a higher seroprevalence for Icterohaemorragiae in 2008 (P=0.003) and Hardjo in 2009 (P=0.041). Risk of exposure to Leptospira was higher at F-sites compared to H-sites (odds ratio 2.3, 95% confidence interval 1.3-3.9, P=0.002). We captured more animals with titers >1:800 at H-sites, but there was no association between titer levels and capture site. Six of eight water sources were Leptospira-positive; however, there was no correlation between trapping locations of seropositive animals and positive water sources. Natural areas create opportunities for interspecies interactions, favoring leptospires transmission across species. Understanding that Leptospira serovars are present in natural areas is an integral part of the safe human and pet recreational use of these areas. Our study should raise awareness and build on public education designed to prevent disease transmission between species.

Carriage of Methicillin-resistant Staphylococcus aureus in a Colony of Rhesus (Macaca mulatta) and Cynomolgus (Macaca fascicularis) Macaques.

Methicillin-resistant Staphylococcus aureus (MRSA) carriage and infection are well documented in the human and veterinary literature; however only limited information is available regarding MRSA carriage and infection in laboratory NHP populations. The objective of this study was to characterize MRSA carriage in a representative research colony of rhesus and cynomolgus macaques through a cross-sectional analysis of 300 animals. MRSA carriage was determined by using nasal culture. Demographic characteristics of carriers and noncarriers were compared to determine factors linked to increased risk of carriage, and MRSA isolates were analyzed to determine antimicrobial susceptibility patterns, staphylococcal chromosome cassette mec (SCCmec) type, and multilocus sequence type (ST). Culture results demonstrated MRSA carriage in 6.3% of the study population. Animals with greater numbers of veterinary or experimental interventions including antibiotic administration, steroid administration, dental procedures, and surgery were more likely to carry MRSA. Susceptibility results indicated that MRSA isolates were resistant to ß-lactams, and all isolates were resistant to between 1 and 4 non ß-lactam antibiotics. In addition, 73.7% of MRSA isolates were identified as ST188-SCCmec IV, an isolate previously observed in an unrelated population of macaques and 15.8% were ST3268-SCCmec V, which has only been described in macaques. A single isolate had a novel sequence type, ST3478, and carried SCCmec V. These results suggest that NHP-adapted strains of MRSA exist and highlight the emergence of antimicrobial resistance in laboratory NHP populations.

Immunomodulatory effects of whole yeast cells and capsicum in weanling pigs challenged with pathogenic Escherichia coli1.

An experiment was conducted to evaluate growth performance, fecal bacterial counts, frequency of diarrhea, and clinical blood parameters in weanling pigs inoculated with enterotoxigenic Escherichia coli (ETEC) who were fed a whole yeast cell (WYC) product and capsicum, a plant essential oil. Weanling pigs (34 barrows and 30 gilts, 21 d of age, 5.90 ± 1.03 kg BW) were allotted to experimental treatments in a randomized complete block design based on litter, sex, and initial BW. Four pigs were individually housed within each containment chamber and assigned to 1 of 4 dietary treatments, which included a control diet without or with 0.2% WYC (CitriStim; ADM, Decatur, IL) or 10 ppm of capsicum (XTract 6933; Pancosma, Geneva, Switzerland), provided either alone or in combination. After receiving diets for 13 d, pigs were orally inoculated with F18+ ETEC and maintained on their assigned diets for an additional 10 d; a separate cohort of 12 pigs receiving the control diet was sham-inoculated using PBS. Body and feeder weights were recorded, and fecal swabs collected, on 0, 5, and 10 d postinoculation (DPI), with blood sampled at 0, 2, 7, and 10 DPI for isolation of peripheral blood mononuclear cells. Pigs challenged with ETEC and fed diets containing WYC or capsicum alone had a higher frequency of diarrhea when compared with pigs receiving diets without those compounds (P < 0.05). Total fecal bacterial counts in pigs fed the combination of additives were highest when compared with either additive alone (interaction, P = 0.03) at 10 DPI. Blood leukocyte counts were increased in challenged pigs receiving the combination of additives compared with all other challenged treatment groups (interaction, P = 0.04). The addition of WYC increased (main effect, P = 0.01) lymphocyte counts at 7 DPI. Proportions of CD8+ and CD4+CD8+ cells were lower in pigs fed the combination of additives compared with pigs fed either additive alone at 0 and 7 DPI. In conclusion, these data indicate that the combination of the 2 additives elicited higher ETEC shedding and circulating leukocyte counts, and reduced the proportions of cytotoxic and memory T-cells than either additive alone.

Emydomyces testavorans, a New Genus and Species of Onygenalean Fungus Isolated from Shell Lesions of Freshwater Aquatic Turtles.

The fungal order Onygenales includes many pathogens of humans and animals, and recent studies have shown some onygenalean fungi to be significant emerging pathogens of reptiles. Although many of these fungi have similar morphological features in histologic tissue sections, recent molecular analyses have revealed a genetically complex and diverse group of reptile pathogens comprising several genera, most notably Nannizziopsis, Ophidiomyces, and Paranannizziopsis Infections by members of these genera have been previously reported in a variety of reptile species, including crocodilians, lizards, snakes, and tuataras, with negative impacts on conservation efforts for some reptiles. Despite the well-documented pathogenicity of these fungi in all other extant reptile lineages, infection has not yet been reported in aquatic turtles. In this study, we report the isolation of an onygenalean fungus associated with shell lesions in freshwater aquatic turtles. The morphologic and genetic characteristics of multiple isolates (n = 21) are described and illustrated. Based on these features and results of a multigene phylogenetic analysis, a new genus and species, Emydomyces testavorans, are proposed for these fungi isolated from turtle shell lesions.

Identification of Cytauxzoon felis infection in domestic cats from southern Illinois.

OBJECTIVES: The objective of this study was to document Cytauxzoon felis infection in domestic cats from southern Illinois. METHODS: Diagnosis of cytauxzoonosis was based upon clinical signs of illness and detection of piroplasms within erythrocytes on peripheral blood smears or schizonts in internal organs consistent with Cytauxzoon infection. Additionally, genomic DNA was extracted from histologic sections of splenic tissue from two cats. RESULTS: The internal transcribed spacer region-1 (ITS-1) and ITS-2 of the C felis genome were successfully sequenced, confirming infection with the organism. CONCLUSIONS AND RELEVANCE: Sequence analysis of C felis DNA isolated from histologic lesions in two domestic cats from southern Illinois show either mixed infection or possible heterozygosity (cytosine and thymine) in ITS-2 at the position equivalent to nucleotide 76 (thymine) in the most commonly isolated C felis ITS-2 sequence. Identification of C felis infection in domestic cats from southern Illinois is a critical finding that raises awareness of this often fatal disease process in an area of the USA where, previously, the disease was only anecdotally reported.