Enhancing the one health initiative by using whole genome sequencing to monitor antimicrobial resistance of animal pathogens: Vet-LIRN collaborative project with veterinary diagnostic laboratories in United States and Canada.

BACKGROUND: Antimicrobial resistance (AMR) of bacterial pathogens is an emerging public health threat. This threat extends to pets as it also compromises our ability to treat their infections. Surveillance programs in the United States have traditionally focused on collecting data from food animals, foods, and people. The Veterinary Laboratory Investigation and Response Network (Vet-LIRN), a national network of 45 veterinary diagnostic laboratories, tested the antimicrobial susceptibility of clinically relevant bacterial isolates from animals, with companion animal species represented for the first time in a monitoring program. During 2017, we systematically collected and tested 1968 isolates. To identify genetic determinants associated with AMR and the potential genetic relatedness of animal and human strains, whole genome sequencing (WGS) was performed on 192 isolates: 69 Salmonella enterica (all animal sources), 63 Escherichia coli (dogs), and 60 Staphylococcus pseudintermedius (dogs). RESULTS: We found that most Salmonella isolates (46/69, 67%) had no known resistance genes. Several isolates from both food and companion animals, however, showed genetic relatedness to isolates from humans. For pathogenic E. coli, no resistance genes were identified in 60% (38/63) of the isolates. Diverse resistance patterns were observed, and one of the isolates had predicted resistance to fluoroquinolones and cephalosporins, important antibiotics in human and veterinary medicine. For S. pseudintermedius, we observed a bimodal distribution of resistance genes, with some isolates having a diverse array of resistance mechanisms, including the mecA gene (19/60, 32%). CONCLUSION: The findings from this study highlight the critical importance of veterinary diagnostic laboratory data as part of any national antimicrobial resistance surveillance program. The finding of some highly resistant bacteria from companion animals, and the observation of isolates related to those isolated from humans demonstrates the public health significance of incorporating companion animal data into surveillance systems. Vet-LIRN will continue to build the infrastructure to collect the data necessary to perform surveillance of resistant bacteria as part of fulfilling its mission to advance human and animal health. A One Health approach to AMR surveillance programs is crucial and must include data from humans, animals, and environmental sources to be effective.

An animal model of a newly emerging human ehrlichiosis.

BACKGROUND: Human ehrlichioses are emerging life-threatening diseases transmitted by ticks. Animal models have been developed to study disease development; however, there is no valid small animal model that uses a human ehrlichial pathogen. The objective of this study was to develop a mouse model for ehrlichiosis with the newly discovered human pathogen, Ehrlichia muris-like agent (EMLA). METHODS: Three strains of mice were inoculated with different doses of EMLA by the intravenous, intraperitoneal, or intradermal route and evaluated for clinical and pathologic changes during the course of infection. RESULTS: EMLA infected C57Bl/6, BALB/c, and C3H/HeN mice and induced lethal or persistent infection in a route- and dose-dependent manner. The clinical chemistry and hematologic changes were similar to those of human infection by Ehrlichia chaffeensis or EMLA. Bacterial distribution in tissues differed after intradermal infection, compared with the distribution after intravenous or intraperitoneal injection. Lethal infection did not cause remarkable pathologic changes, but it caused fluid imbalance. EMLA infection of endothelium and mononuclear cells likely plays a role in the severe outcome. CONCLUSIONS: The EMLA mouse model mimics human infection and can be used to study pathogenesis and immunity and for development of a vector transmission model of ehrlichiosis.

Early induction of interleukin-10 limits antigen-specific CD4⁺ T cell expansion, function, and secondary recall responses during persistent phagosomal infection.

Diverse pathogens have evolved to survive and replicate in the endosomes or phagosomes of the host cells and establish persistent infection. Ehrlichiae are Gram-negative, intracellular bacteria that are transmitted by ticks. Ehrlichiae reside in the endosomes of the host phagocytic or endothelial cells and establish persistent infection in their vertebrate reservoir hosts. CD4(+) T cells play a critical role in protection against phagosomal infections. In the present study, we investigated the expansion, maintenance, and functional status of antigen-specific CD4(+) T cells during persistent Ehrlichia muris infection in wild-type and interleukin-10 (IL-10)-deficient mice. Our study indicated that early induction of IL-10 led to reduced inflammatory responses and impaired bacterial clearance during persistent Ehrlichia infection. Notably, we demonstrated that the functional production of gamma interferon (IFN-γ) by antigen-specific CD4(+) T cells maintained during a persistent phagosomal infection progressively deteriorates. The functional loss of IFN-γ production by antigen-specific CD4(+) T cells was reversed in the absence of IL-10. Furthermore, we demonstrated that transient blockade of IL-10 receptor during the T cell priming phase early in infection was sufficient to enhance the magnitude and the functional capacity of antigen-specific effector and memory CD4(+) T cells, which translated into an enhanced recall response. Our findings provide new insights into the functional status of antigen-specific CD4(+) T cells maintained during persistent phagosomal infection. The study supports the concept that a better understanding of the factors that influence the priming and differentiation of CD4(+) T cells may provide a basis to induce a protective immune response against persistent infections.

Improved molecular detection of Neorickettsia risticii with a duplex real-time PCR assay in the diagnosis of Potomac horse fever.

Neorickettsia risticii, an obligate intracellular bacterium, is the causative agent of Potomac horse fever (PHF). Diagnosis of PHF is based on demonstration of serum antibodies, isolation of N. risticii, and/or detection of nucleic acid by a PCR assay. An existing real-time PCR assay targeting the N. risticii 16S rRNA has been validated using blood samples from horses with colitis, and snails; to our knowledge, the performance of the assay for other sample types has not been reported. We describe here a modification of the 16S rRNA gene assay by the addition of a set of primers and probe targeting the N. risticii p51 gene to form a duplex assay. We validated the new assay using diagnostic specimens from 56 horses with suspected PHF. The assay consistently detected down to 5 copies of synthetic targets, and did not show any cross-reaction with common equine enteric pathogens. Although we did not establish the diagnostic sensitivity and specificity of the duplex assay, results for both gene targets were in complete agreement, with the exception of 4 fecal samples that tested positive for the 16S rRNA gene only. Further analysis indicated that testing of fecal samples using our 16S rRNA gene assay alone can produce a false-positive result.

SARS-CoV-2 Infection Dynamics in the Pittsburgh Zoo Wild Felids with Two Viral Variants (Delta and Alpha) during the 2021-2022 Pandemic in the United States.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been reported in multiple animal species besides humans. The goal of this study was to report clinical signs, infection progression, virus detection and antibody response in a group of wild felids housed in adjacent but neighboring areas at the Pittsburgh Zoo. Initially, five African lions (Panthera leo krugeri) housed together exhibited respiratory clinical signs with viral shedding in their feces in March of 2021 coinciding with infection of an animal keeper. During the second infection wave in December 2021, four Amur tigers (Panthera tigris altaica) and a Canadian lynx (Lynx canadensis) showed clinical signs and tested positive for viral RNA in feces. In infected animals, viral shedding in feces was variable lasting up to 5 weeks and clinical signs were observed for up to 4 weeks. Despite mounting an antibody response to initial exposure, lions exhibited respiratory clinical signs during the second infection wave, but none shed the virus in their feces. The lions were positive for alpha variant (B.1.1.7 lineage) during the first wave and the tiger and lynx were positive for delta variant (AY.25.1. lineage) during the second wave. The viruses recovered from felids were closely related to variants circulating in human populations at the time of the infection. Cheetahs (Acinonyx jubatus) in the park did not show either the clinical signs or the antibody response.

Prevalence of Anaplasma phagocytophilum and Borrelia burgdorferi infection in horses from Pennsylvania (2017-2019) using antibody and organism-based detection.

OBJECTIVE: To determine the prevalence of Anaplasma phagocytophilum and Borrelia burgdorferi infections in Pennsylvania horses. ANIMALS: 271 horses. PROCEDURES: A survey was conducted with PCR and serology to evaluate anaplasmosis and Lyme disease infections in horses from Pennsylvania that were suspected for tick-borne infection. RESULTS: A phagocytophilum was detected in 19/271 (7.0%) Pennsylvania horses tested by the duplex PCR. B burgdorferi was not detected in any horse blood tested by PCR. Overall, 120/271 (44.3%) horses tested positive for presence of A phagocytophilum antibodies by at least the IDEXX SNAP 4Dx Plus lateral flow immunosorbent (SNAP) or indirect fluorescent antibody (IFA) assay, with 69 (25.5%) testing positive by both SNAP and IFA; 43 (15.9%) tested positive by IFA only, and 8 (3.0%) tested positive by SNAP only. Similarly, 209/271 (77.1%) horses tested positive for the presence of B burgdorferi antibodies by at least 1 test, with 139 (51.3%) testing positive by both SNAP and IFA; 45 (16.6%) tested positive by SNAP only, and 25 (9.2%) tested positive by IFA. CLINICAL RELEVANCE: Both A phagocytophilum and B burgdorferi are important tick-borne infections. The study provides prevalence data for both A phagocytophilum and B burgdorferi and compares test performance. For serologic detection, IFA detected antibodies to A phagocytophilum in a higher proportion (41.3%) of horses compared to SNAP (28.4%), while SNAP detected antibodies to B burgdorferi in a higher proportion (67.9%) of horses compared to IFA (60.5%). Both diseases showed a high seroprevalence in all areas surveyed.

An unusual outbreak of erysipelas on a goat farm in Pennsylvania.

Erysipelothrix rhusiopathiae infection and septicemia occurred in a 5-d old Boer goat found dead on a farm in western Pennsylvania. On autopsy, there was moderate, focally extensive hemorrhage along the remnants of the urachus and umbilical arteries and the apex of the urinary bladder. Microscopic examination of immunohistochemical stained tissues revealed abundant intracellular and extracellular E. rhusiopathiae antigen-positive bacilli in all tissues stained, including lung, heart, liver, skeletal muscle, kidney, and thymus. Bacteria isolated from liver and urachus were identified as E. rhusiopathiae by MALDI-TOF mass spectrometry and further confirmed by a PCR assay. An epidemiologic investigation was conducted via an on-farm questionnaire after the owners noted a 70% mortality rate from the 2019 kidding season. The epidemiologic investigation showed that E. rhusiopathiae, an opportunistic zoonotic organism, was introduced to the farm through a breach in biosecurity and was likely perpetuated among the resident poultry species.

Transmission of SARS-CoV-2 from humans to a 16-year-old domestic cat with comorbidities in Pennsylvania, USA.

BACKGROUND AND OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), besides causing human infection, has been shown to naturally infect several susceptible animal species including large cats (tigers, lions, pumas, spotted leopards), dogs, cats, ferrets, gorillas and minks. Cats and minks are continuing to be the most reported species with SARS-CoV-2 infections among animals but it needs to be investigated further. METHODS AND RESULTS: We report the detection of SARS-CoV-2 from a domestic cat that exhibited respiratory disease after being exposed to SARS-CoV-2 virus from humans in the same household. SARS-CoV-2 RNA was detected in two oropharyngeal swabs collected at two time points, 11 days apart; the first, when the cat was reported to be sick and the second, before euthanasia due to poor prognosis. The viral nucleic acid detected at two time points showed no genomic variation and resembled the clade GH circulating in humans in the United States. Clinical and pathological findings noted in this 16-year-old cat were consistent with respiratory and cardiac insufficiency. CONCLUSIONS: SARS-CoV-2 viral infection was likely an incidental clinical finding, as the virus was not detected in fixed lungs, heart, or kidney tissues. Only fresh lung tissue collected at necropsy showed the presence of viral nucleic acid, albeit at a very low level. Further research is needed to clarify the clinical course of SARS-CoV-2 in companion animals of advanced age and underlying cardiac disease.

Natural killer cells promote tissue injury and systemic inflammatory responses during fatal Ehrlichia-induced toxic shock-like syndrome.

Human monocytotropic ehrlichiosis is caused by Ehrlichia chaffeensis, a Gram-negative bacterium lacking lipopolysaccharide. We have shown that fatal murine ehrlichiosis is associated with CD8(+)T cell-mediated tissue damage, tumor necrosis factor-alpha, and interleukin (IL)-10 overproduction, and CD4(+)Th1 hyporesponsiveness. In this study, we examined the relative contributions of natural killer (NK) and NKT cells in Ehrlichia-induced toxic shock. Lethal ehrlichial infection in wild-type mice induced a decline in NKT cell numbers, and late expansion and migration of activated NK cells to the liver, a main infection site that coincided with development of hepatic injury. The spatial and temporal changes in NK and NKT cells in lethally infected mice correlated with higher NK cell cytotoxic activity, higher expression of cytotoxic molecules such as granzyme B, higher production of interferon-gamma and tumor necrosis factor-alpha, increased hepatic infiltration with CD8alphaCD11c(+) dendritic cells and CD8(+)T cells, decreased splenic CD4(+)T cells, increased serum concentrations of IL-12p40, IL-18, RANTES, and monocyte chemotactic protein-1, and elevated production of IL-18 by liver mononuclear cells compared with nonlethally infected mice. Depletion of NK cells prevented development of severe liver injury, decreased serum levels of interferon-gamma, tumor necrosis factor-alpha, and IL-10, and enhanced bacterial elimination. These data indicate that NK cells promote immunopathology and defective anti-ehrlichial immunity, possibly via decreasing the protective immune response mediated by interferon-gamma producing CD4(+)Th1 and NKT cells.

Evaluation of a high-throughput nucleic acid extraction method for the detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples by PCR.

Johne's disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.

Detection of Anaplasma Phagocytophilum in Horses With Suspected Tick-Borne Disease in Northeastern United States by Metagenomic Sequencing.

Metagenomic sequencing of clinical diagnostic specimens has a potential for unbiased detection of infectious agents, diagnosis of polymicrobial infections and discovery of emerging pathogens. Herein, next generation sequencing (NGS)-based metagenomic approach was used to investigate the cause of illness in a subset of horses recruited for a tick-borne disease surveillance study during 2017-2019. Blood samples collected from 10 horses with suspected tick-borne infection and five apparently healthy horses were subjected to metagenomic analysis. Total genomic DNA extracted from the blood samples were enriched for microbial DNA and subjected to shotgun next generation sequencing using Nextera DNA Flex library preparation kit and V2 chemistry sequencing kit on the Illumina MiSeq sequencing platform. Overall, 0.4-0.6 million reads per sample were analyzed using Kraken metagenomic sequence classification program. The taxonomic classification of the reads indicated that bacterial genomes were overrepresented (0.5 to 1%) among the total microbial reads. Most of the bacterial reads (~91%) belonged to phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria and Tenericutes in both groups. Importantly, 10-42.5% of Alphaproteobacterial reads in 5 of 10 animals with suspected tick-borne infection were identified as Anaplasma phagocytophilum. Of the 5 animals positive for A. phagocytophilum sequence reads, four animals tested A. phagocytophilum positive by PCR. Two animals with suspected tick-borne infection and A. phagocytophilum positive by PCR were found negative for any tick-borne microbial reads by metagenomic analysis. The present study demonstrates the usefulness of the NGS-based metagenomic analysis approach for the detection of blood-borne microbes.

A Novel Real-Time PCR Assay for the Rapid Detection of Virulent Streptococcus equi Subspecies zooepidemicus-An Emerging Pathogen of Swine.

Streptococcus equi subspecies zooepidemicus, a zoonotic bacterial pathogen caused a series of outbreaks with high mortality affecting swine herds in multiple locations of the USA and Canada in 2019. Further genetic analysis revealed that this agent clustered with ATCC 35246, a S. zooepidemicus strain associated with high mortality outbreaks in swine herds of China originally reported in 1977. Rapid and accurate diagnosis is absolutely critical for controlling and limiting further spread of this emerging disease of swine. Currently available diagnostic methods including bacteriological examination and PCR assays do not distinguish between the virulent strains and avirulent commensal strains of S. zooepidemicus, which is critical given that this pathogen is a normal inhabitant of the swine respiratory tract. Based on comparative analyses of whole genome sequences of the virulent isolates and avirulent sequences, we identified a region in the SzM gene that is highly conserved and restricted to virulent S. zooepidemicus strains. We developed and validated a novel probe-based real-time PCR targeting the conserved region of SzM. The assay was highly sensitive and specific to the virulent swine isolates of Streptococcus equi subspecies zooepidemicus. No cross reactivity was observed with avirulent S. zooepidemicus isolates as well as other streptococcal species and a panel of porcine respiratory bacterial and viral pathogens. The PCR efficiency of the assay was 96.64 % and was able to detect as little as 20 fg of the bacterial DNA. We then validated the diagnostic sensitivity and specificity of the new PCR assay using a panel of clinical samples (n = 57) and found that the assay has 100% sensitivity and specificity as compared to bacteriological culture method. In summary, the PCR assay will be an extremely valuable tool for the rapid accurate detection of virulent swine S. zooepidemicus isolates and directly from clinical samples.

Detection of Babesia, Borrelia, Anaplasma, and Rickettsia spp. in Adult Black-Legged Ticks (Ixodes scapularis) from Pennsylvania, United States, with a Luminex Multiplex Bead Assay.

Ixodes scapularis, the black-legged tick, harbors multiple organisms and transmits several pathogens to animals and humans. To determine the presence of tick-borne microorganisms carried by I. scapularis in Pennsylvania, 299 adult I. scapularis ticks were collected from across the state and tested with a multiplex bead panel targeting 20 microorganisms. The Luminex bead-based xMAP® MultiFLEX Mega Tick Panel detected microorganisms in these ticks, including Anaplasma spp. (1.7%), Borrelia spp. (45.8%), Babesia spp. (16.1%), and Rickettsia spp. (22.1%) at the genera level and identified Anaplasma phagocytophilum (1.7%), Babesia microti (0.7%), Borrelia burgdorferi sensu stricto (45.5%), Borrelia miyamotoi (0.3%), and Rickettsia parkeri (0.7%) at the species level. Babesia spp. reactivity was found to be due to Ba. odocoilei, and Rickettsia spp. reactivity was mainly due to rickettsial endosymbionts.

Draft Genome Sequences of Two Virulent Streptococcus equi subsp. zooepidemicus Swine Isolates from Pennsylvania.

Draft genome sequences of two outbreak isolates of Streptococcus equi subsp. zooepidemicus from a Pennsylvania swine herd affected with high mortality and morbidity are reported here. The genome analysis revealed that the isolates are closely related to a virulent strain originally identified in China.

Persistent infection contributes to heterologous protective immunity against fatal ehrlichiosis.

Human monocytotropic ehrlichiosis (HME), an emerging and often life-threatening tick-transmitted disease, is caused by the obligately intracellular bacterium Ehrlichia chaffeensis. HME is modeled in C57BL/6 mice using Ehrlichia muris, which causes persistent infection, and Ixodes ovatus Ehrlichia (IOE), which is either acutely lethal or sublethal depending on the dose and route of inoculation. A persistent primary E. muris infection, but not a sublethal IOE infection, protects mice against an ordinarily lethal secondary IOE challenge. In the present study, we determined the role of persistent infection in maintenance of protective memory immune responses. E. muris-infected mice were treated with doxycycline or left untreated and then challenged with an ordinarily lethal dose of IOE. Compared to E. muris-primed mice treated with doxycycline, untreated mice persistently infected with E. muris had significantly greater numbers of antigen-specific gamma interferon-producing splenic memory T cells, significant expansion of CD4(+) CD25(+) T regulatory cells, and production of transforming growth factor beta1 in the spleen. Importantly, E. muris-primed mice treated with doxycycline showed significantly greater susceptibility to challenge infection with IOE compared to untreated mice persistently infected with E. muris. The study indicated that persistent ehrlichial infection contributes to heterologous protection by stimulating the maintenance of memory T-cell responses.

A probe-based real-time PCR assay for the detection of Neospora caninum in clinical samples from cattle.

Neospora caninum is an apicomplexan protozoan parasite that is a leading cause of abortion in cattle. Detection of parasite-specific DNA by PCR is a highly sensitive method for identifying the presence of N. caninum in a variety of tissues. We developed and validated a probe-based real-time PCR assay targeting the conserved Nc5 gene of N. caninum. Using N. caninum strain Nc-1 genomic DNA and a synthetic gene fragment as amplification standards, we determined the PCR amplification efficiency and the limit of detection to be 95.60% and 3 copies, respectively. Five pathogens frequently associated with bovine abortions, namely bovine viral diarrhea virus types I and II, bovine alphaherpesvirus-1, Chlamydia, and Leptospira, were tested to ensure analytical exclusivity. A total of 103 clinical samples from aborted fetuses were tested concurrently with a standard conventional PCR and the new probe-based real-time PCR assay. All tested samples showed 100% agreement between these two assays. In conclusion, the probe-based real-time PCR assay facilitates accurate and rapid detection of N. caninum from abortions in cattle.

Protective heterologous immunity against fatal ehrlichiosis and lack of protection following homologous challenge.

The roles of antibodies and memory T cells in protection against virulent Ehrlichia have not been completely investigated. In this study, we addressed these issues by using murine models of mild and fatal ehrlichiosis caused by related monocytotropic Ehrlichia strains. Mice were primed with either Ehrlichia muris or closely related virulent ehrlichiae transmitted by Ixodes ovatus (IOE) ticks given intraperitoneally or intradermally. All groups were reinfected intraperitoneally, 30 days later, with a lethal high dose of IOE. Priming with E. muris, but not IOE, induced strong CD4+ and CD8+ memory type 1 T-cell responses, Ehrlichia-specific immunoglobulin G (IgG) antibodies, and persistent infection. Compared to IOE-primed mice, subsequent lethal IOE challenge of E. muris-primed mice, resulted in (i) 100% protection against lethal infection, (ii) strong Ehrlichia-specific secondary gamma interferon (IFN-gamma)-producing effector/effector memory CD4+ and CD8+ T-cell responses, (iii) enhanced secondary anti-ehrlichial antibody response, (iv) accelerated bacterial clearance, and (v) the formation of granulomas in the liver and lung. E. muris-primed mice challenged with IOE had lower levels of serum interleukin-1alpha (IL-1alpha), IL-6, and IL-10 compared to unprimed mice challenged with IOE. Interestingly, the fatal secondary response in IOE-primed mice correlated with (i) decline in the Ehrlichia-specific CD4+ and CD8+ type 1 responses, (ii) marked hepatic apoptosis and necrosis, and (iii) substantial bacterial clearance, suggesting that fatal secondary response is due to immune-mediated tissue damage. In conclusion, protection against fatal ehrlichial infection correlates with strong expansion of IFN-gamma-producing CD4+ and CD8+ effector memory type 1 T cells, which appear to be maintained in the presence of IgG antibodies and persistent infection.

Comparison of three DNA extraction methods for molecular confirmation of Mycobacterium avium subspecies paratuberculosis from the VersaTrek™ liquid cultures of bovine fecal samples.

We evaluated three DNA extraction methods for confirmation of Mycobacterium avium subspecies paratuberculosis from liquid cultures of bovine feces. Use of DNA Extract All Reagents Kit™ resulted in efficient extraction of amplifiable DNA from higher proportion (96.29%) of known positive samples compared to Chelex-100 resin (25.92%) and polyethylene glycol (0%).

Detection of Brucella canis infection in dogs by blood culture and bacterial identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Brucella canis was recovered from dogs that were canine brucellosis suspect by blood culture using a modified lysis method. Organism identity was established by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The instrument-provided security library identified the isolates as Brucella species. The isolates were further identified as B. canis with the help of phenotypic and genotypic characteristics. The mass spectral profiles from characterized B. canis isolates, when added to the MALDI-TOF MS standard reference library, allowed successful presumptive identification of B. canis.

Bovine cumulus-granulosa cells contain biologically active retinoid receptors that can respond to retinoic acid.

Retinoids, a class of compounds that include retinol and its metabolite, retinoic acid, are absolutely essential for ovarian steroid production, oocyte maturation, and early embryogenesis. Previous studies have detected high concentrations of retinol in bovine large follicles. Further, administration of retinol in vivo and supplementation of retinoic acid during in vitro maturation results in enhanced embryonic development. In the present study, we hypothesized that retinoids administered either in vivo previously or in vitro can exert receptor-mediated effects in cumulus-granulosa cells. Total RNA extracted from in vitro cultured cumulus-granulosa cells was subjected to reverse transcription polymerase chain reaction (RT-PCR) and mRNA expression for retinol binding protein (RBP), retinoic acid receptor alpha (RARalpha), retinoic acid receptor beta (RARbeta), retinoic acid receptor gamma (RARgamma), retinoid X receptor alpha (RXRalpha), retinoid X receptor beta (RXRbeta), retinaldehyde dehydrogenase-2 (RALDH-2), and peroxisome proliferator activated receptor gamma (PPARgamma). Transcripts were detected for RBP, RARalpha, RARgamma, RXRalpha, RXRbeta, RALDH-2, and PPARgamma. Expression of RARbeta was not detected in cumulus-granulosa cells. Using western blotting, immunoreactive RARalpha, and RXRbeta protein was also detected in bovine cumulus-granulosa cells. The biological activity of these endogenous retinoid receptors was tested using a transient reporter assay using the pAAV-MCS-betaRARE-Luc vector. Addition of 0.5 and 1 micro molar all-trans retinoic acid significantly (P < 0.05) increased the activity of the pAAV-MCS-betaRARE-Luc reporter compared to cells transfected with the control reporter lacking a retinoic acid response element. Addition of 5 or 10 micro molar all-trans retinol stimulated a mild increase in reporter activity, however, the increase was not statistically significant. Based on these results we conclude that cumulus cells contain endogenously active retinoid receptors and may also be competent to synthesize retinoic acid using the precursor, retinol. These results also indirectly provide evidence that retinoids administered either in vivo previously or in vitro may have exerted a receptor-mediated effect on cumulus-granulosa cells.