Genomic epidemiology of third-generation cephalosporin-resistant Escherichia coli from Argentinian pig and dairy farms reveals animal-specific patterns of co-resistance and resistance mechanisms.

Control measures are being introduced globally to reduce the prevalence of antibiotic resistance (ABR) in bacteria on farms. However, little is known about the current prevalence and molecular ecology of ABR in bacterial species with the potential to be key opportunistic human pathogens, such as Escherichia coli, on South American farms. Working with 30 dairy cattle farms and 40 pig farms across two provinces in central-eastern Argentina, we report a comprehensive genomic analysis of third-generation cephalosporin-resistant (3GC-R) E. coli, which were recovered from 34.8% (cattle) and 47.8% (pigs) of samples from fecally contaminated sites. Phylogenetic analysis revealed substantial diversity suggestive of long-term horizontal and vertical transmission of 3GC-R mechanisms. CTX-M-15 and CTX-M-2 were more often produced by isolates from dairy farms, while CTX-M-8 and CMY-2 and co-carriage of amoxicillin/clavulanate resistance and florfenicol resistance were more common in isolates from pig farms. This suggests different selective pressures for antibiotic use in these two animal types. We identified the ß-lactamase gene blaROB, which has previously only been reported in the family Pasteurellaceae, in 3GC-R E. coli. blaROB was found alongside a novel florfenicol resistance gene, ydhC, also mobilized from a pig pathogen as part of a new composite transposon. As the first comprehensive genomic survey of 3GC-R E. coli in Argentina, these data set a baseline from which to measure the effects of interventions aimed at reducing on-farm ABR and provide an opportunity to investigate the zoonotic transmission of resistant bacteria in this region. IMPORTANCE: Little is known about the ecology of critically important antibiotic resistance among bacteria with the potential to be opportunistic human pathogens (e.g., Escherichia coli) on South American farms. By studying 70 pig and dairy cattle farms in central-eastern Argentina, we identified that third-generation cephalosporin resistance (3GC-R) in E. coli was mediated by mechanisms seen more often in certain species and that 3GC-R pig E. coli were more likely to be co-resistant to florfenicol and amoxicillin/clavulanate. This suggests that on-farm antibiotic usage is key to selecting the types of E. coli present on these farms. 3GC-R E. coli and 3GC-R plasmids were diverse, suggestive of long-term circulation in this region. We identified the de novo mobilization of the resistance gene blaROB from pig pathogens into E. coli on a novel mobile genetic element, which shows the importance of surveying poorly studied regions for antibiotic resistance that might impact human health.

Detection of nonpregnant cows and potential embryo losses by color Doppler ultrasound and interferon-stimulated gene expression in grazing dairy cows.

Many studies have been conducted to estimate pregnancy losses between 19 and 34 d after artificial insemination (AI) in dairy cows managed under confinement-based systems, but few studies have examined embryo mortality during this interval in dairy cows managed under gazing systems. The objectives of this prospective cohort study were (1) to assess the diagnostic value of the corpus luteum (CL) blood perfusion (BP) evaluation by Doppler ultrasound (US) to detect nonpregnant cows at 19 to 20 d post-AI, and (2) to assess the rate of potential embryo mortality between 19 to 34 d post-AI. The CL-BP of all cows included in the study (n = 131) was examined on farm by power and color mode of Doppler US and later using an image processing software by a second evaluator. The endometrium thickness and echotexture were evaluated by B-mode US at the same visit to assess if the nonpregnancy diagnosis could be improved at 19 to 20 d post-AI by this additional diagnostic tool. Blood samples were obtained at 19 to 20 d post-AI for progesterone (P4) measurement by chemiluminescence and to determine the mRNA expression of ISG by real-time PCR. Pregnancy diagnosis based on embryo visualization was performed at 33 to 34 d post-AI by US B-mode. In parallel interpretation, ISG15 and MX2 mRNA expression in leukocytes [sensitivity (Se), 100%] were regarded as suitable biomarkers for early pregnancy and were selected for molecular characterization of pregnancy at 19 to 20 d post-AI. At 19 to 20 d post-AI, 61.1% of the cows had positive CL-BP by Doppler US (Se, 98.0%), 62.7% had ISG mRNA expression in leukocytes over the cutoff point (Se, 95.7%), and 50.8% were positive, based on the combination of ISG mRNA expression, CL-BP by Doppler US, and P4 concentration (Se, 100%), and were considered as possible pregnant. At 33 to 34 d, the pregnancy rate was 37.4% diagnosed by the B-mode US. Based on the expression of the selected biomarkers in cows with active CL, we found that 28.1% of the cows could have potentially lost their pregnancy between 19 and 34 d post-AI. The Doppler US color mode showed similar accuracy and a higher negative predictive value than the genes selected as biomarkers. The additional B-mode ultrasound evaluation of the uterine stratum vasculare and the endometrium thickness improved the diagnostic accuracy. Therefore, assessing the CL-BP by Doppler US allowed early detection of nonpregnant cows at 19 to 20 d post-AI. The combination of early CL-BP by Doppler US (d 19 to 20) with early embryo detection by B-mode US (d 33-34) could be used to facilitate earlier rebreeding of dairy cows.

Association between progesterone concentration and endometrial gene expression in dairy cows.

The objective of this study was to evaluate the association between progesterone concentration on Days 4 and 9 of the estrus cycle and endometrial transcriptome at Day 9 in lactating grazing dairy cows. Blood samples were obtained on Days 0, 4, and 9 for progesterone measurement by chemiluminescence. Cows were assigned to one of the following groups (n = 3 per group): cows with low physiological progesterone on Day 4, cows in anestrous, cows with high physiological progesterone on Day 4, and superovulated cows. Endometrial biopsy samples were obtained on Day 9 for RNA sequencing. Quality control and determination of differentially expressed genes (false discovery rate <0.05) were determined using the edgeR package for R software. We identified 3,042 differentially expressed genes among the 4 groups. Cows having high physiological progesterone and superovulated cows showed high similarities and clustered apart from those in anestrus or having low physiological progesterone. Functional analysis using Database for Annotation, Visualization, and Integrated Discovery revealed that endometrial genes upregulated by low progesterone concentration are enriched genes involved in the immune system and inflammatory response. Conversely, cows with high physiological progesterone concentration presented an endometrial transcriptome with similarities to cows with good genetic merit for fertility, showing upregulation of genes related to uterine relaxation-contraction, focal adhesion, GnRH signaling pathway, and epidermal growth factor-like related terms, suggesting a favorable embryo environment. In conclusion, our results support the concept that there is a threshold of progesterone concentration at the beginning of the luteal phase associated with endometrial expression of critical genes involved in the preparation of the uterine environment for embryo implantation.

Short communication: Repeat breeder cows with fluid in the uterine lumen had poorer fertility.

The objective of the present study was to test the association between the presence of fluid in uterine lumen and the fertility of repeat breeder cows (RBC). Grazing Holstein cows that were clinically normal and were not pregnant after at least 3 consecutive inseminations were defined as RBC and included in the study (n = 358). A cut-point ≥2 mm of uterine lumen visible by ultrasonography was used as indicative of the presence of fluid in the uterus. The effect of this fluid on the fertility of RBC was assessed by mixed logistic regression. Twenty percent of the RBC had ≥2 mm of fluid in their uterus, and this finding was associated with a marked reduction in the odds of conception (odds ratio = 0.46). Additional intrauterine sampling revealed that 24.0% of RBC showed signs of subclinical endometritis [≥5% neutrophils in endometrial cytology (SCE)]. However, we found no agreement between the finding of uterine fluid and the diagnosis of SCE (kappa coefficient = 0.03, 95% confidence interval = -0.07 to 0.14). In conclusion, the presence of fluid (≥2 mm) in uterine lumen, detected by ultrasonography, was associated with lower fertility in RBC, but was not related to the diagnosis of SCE. Thus, ultrasonographic evaluation is an important tool to assist in assessment of RBC.

Application of a bacteriological on-farm test to reduce antimicrobial usage in dairy cows with purulent vaginal discharge.

The objective of this study was to assess the effect of a selective antibiotic treatment strategy based on a quick bacteriological on-farm test (Petrifilm, 3M Corp., St. Paul, MN) compared with the conventional antibiotic treatment of all cows having clinical endometritis (CE) defined by the presence of purulent vaginal discharge on both clinical cure rate and reproductive performance. The study was simultaneously conducted with dairy cows reared under a highly supplemented rotational grazing system in Argentina and in a freestall system in Slovakia. Cows having an abnormal vaginal discharge (VD, indicative of clinical endometritis) on 21 to 35 d in milk (DIM) were randomly allocated to 1 of 2 study groups: selective treatment (ST) or conventional treatment (CT). All cows in the CT group (n = 174) received a single intrauterine administration of 500 mg of cephapirin. In the ST group (n = 178), treatment decision was made according to the results of the bacteriological on-farm test. For this test, we collected intrauterine samples with the cytobrush technique and stroke the brushes onto 2 different Petrifilm plates, one for aerobic count and another for Enterobacteriaceae count, incubated the plates, and counted the number of colonies after 24 h. Positive cows (≥5 colonies in one or both plates) received a single intrauterine treatment with 500 mg of cephapirin, whereas negative cows (<5 colonies) remained untreated. Clinical cure rate was assessed by direct vaginal inspection at 14 d after treatment (VD-0). The odds for conception at first artificial insemination, artificial insemination by 80 DIM, pregnancy by 100 DIM, and for nonpregnancy by 200 DIM were estimated with mixed logistic regression models. The hazard of conception was also assessed with proportional hazard regression model. The selective antibiotic treatment strategy based on the outcome of Petrifilm test reduced the number of required treatments (57%) and maintained similar efficacy in terms of clinical cure and reproductive performance as the conventional antibiotic treatment of all endometritic cows.

Endometrial cytology, biopsy, and bacteriology for the diagnosis of subclinical endometritis in grazing dairy cows.

The objectives of this study were to assess the agreement between endometrial cytology and uterine biopsy for the diagnosis of subclinical endometritis (SEND) in grazing dairy cows, the interobserver agreement of the biopsy's readings, and the bacterial population isolated from the uterus of cows having SEND. In experiment 1, lactating Holstein cows (n=44) 31 to 59 d in milk (DIM) at sampling were enrolled. Clinical endometritis was diagnosed by direct evaluation of vaginal discharge and SEND by endometrial cytology evaluation. Two hundred cells per smear were counted to determine the percentage of polymorphonuclear neutrophilic leukocytes (PMNL). Cut-off values used were ≥8% PMNL at ≤33 DIM, ≥6% PMNL at 34 to 47 DIM, and ≥4% PMNL at ≥48 DIM. Biopsies were assessed blindly by 2 observers who categorized them into 4 groups according to their inflammatory changes: none, minimal, moderate, and severe inflammatory changes. Data were analyzed using the kappa coefficient and logistic regression. In experiment 2, lactating Holstein cows (n=60) 21 to 62 DIM were enrolled. Clinical endometritis and SEND were diagnosed as previously described. Samples were cultured for aerobic and anaerobic bacteria by routine methods of bacteriological testing. Data were analyzed with logistic regression. In experiment 1, little agreement was observed between cytology and biopsy outputs (kappa=0.151), and strong agreement between the 2 operators (kappa=0.854). The likelihood of having a normal biopsy (no inflammatory change) was greater for healthy cows than for those having SEND (odds ratio=13.145). The probability for getting normal uterine tissue decreased 2.1% for every increasing percentage point in PMNL. In experiment 2, no bacteria were isolated from cows with SEND, coagulase-negative staphylococci were commonly isolated from healthy cows, and Trueperella pyogenes was frequently isolated from cows with clinical endometritis. The likelihood of isolating T. pyogenes from uterine samples increased with the percentage of PMNL (odds ratio=1.100). In conclusion, biopsy showed low agreement with cytology for the diagnosis of SEND. Nevertheless, fertility trials using uterine biopsies to predict pregnancy outcomes are needed to determine its diagnostic usefulness. Finally, bacteriology would not be recommended as a diagnostic tool because no bacteria were isolated from cows with SEND.

The relationship between endometrial cytology during estrous cycle and cutoff points for the diagnosis of subclinical endometritis in grazing dairy cows.

The objectives of this study were to assess the effect of the stage of estrous cycle on the percentage of endometrial polymorphonuclear cells (PMN) obtained by cytobrush to determine cutoff values for the diagnosis of subclinical endometritis under pastoral conditions, to measure the prevalence of subclinical endometritis 21 to 62d in milk (DIM), and to evaluate the effect of subclinical endometritis on reproductive performance in grazing dairy cows. The first experiment was conducted on a commercial dairy farm in Buenos Aires province (Argentina), where 17 postpartum cyclic dairy cows without clinical endometritis were selected and synchronized by Ovsynch protocol. Endometrial cytology (cytobrush technique) and blood (tail vessels) samples were obtained on d0, 4, 11, and 18 of the estrous cycle (corresponding to estrus, metestrus, diestrus, and proestrus, respectively) and used for measuring percentage of PMN and P4 concentration, respectively. The percentage of PMN was determined 3times by blinded count by 2 operators. Data were analyzed with PROC MIXED, PROC GENMOD, and PROC FREQ from SAS 9.1. The percentage of PMN did not vary with the stage of the estrous cycle. In addition, PMN counts were below any of the reported thresholds in this study (4%) for most of the cows. Therefore, the risk for false positive test results as a consequence of physiological changes in the counts of PMN during estrous cycle is low. The second experiment was conducted on 4 commercial dairy farms in Buenos Aires province (Argentina), where lactating Holstein dairy cows (n=418) 21 to 62 DIM without clinical endometritis were studied. Samples of endometrial cytology were collected with the cytobrush technique. Data were analyzed with receiver operator characteristic curves with Sigmaplot 10.0, and with PROC GLIMMIX, PROC PHREG, and PROC LIFETEST from SAS 9.1. Cutoff values for the diagnosis of subclinical endometritis in grazing dairy cows are 8% PMN for 21 to 33 DIM, 6% PMN for 34 to 47 DIM, 4% PMN for 48 to 62 DIM, and overall 5% PMN for 21 to 62 DIM; the prevalence of subclinical endometritis 21 to 62 DIM was 17%. Finally, subclinical endometritis diagnosed at 21 to 62 DIM decreases the hazard for pregnancy (hazard ratio=0.668; 95% confidence interval=0.492-0.909) and increases the calving to conception interval by d30 compared with normal cows (median 95% confidence interval=133 vs. 93, respectively).

Prevalence of clinical endometritis and its impact on reproductive performance in grazing dairy cattle in Argentina.

The objective of this study was to evaluate the prevalence of clinical endometritis and its impact on reproductive performance in grazing dairy cattle in Argentina to compare data with previous reports from herds kept in confinement housing systems. A total of 243 Holstein dairy cows from three commercial dairy farms in Buenos Aires Province (Argentina) were examined for the signs of clinical endometritis 18-38 days postpartum (dpp) by external inspection and manual vaginal examination. Vaginal discharge was scored into the categories VDS 0 (transparent, clear mucus), VDS 1 (mucupurulent discharge), VDS 2 (purulent discharge) and VDS 3 (purulent discharge with fetid odour). Cows diagnosed with VDS 1 to VDS 3 were regarded as affected with clinical endometritis and cows with VDS 0 as free of clinical endometritis. All cows were re-examined 14 days later following the same examination protocol. Prevalence of clinical endometritis 18-38 dpp was 35% and decreased to 18% at re-examination. Cows with no palpable ovarian structures or periparturient disorders were at higher risk for clinical endometritis. Hazard for pregnancy was significantly lower in cows with purulent or fetid odour discharge compared with reference cows with no discharge (HR=0.49; p=0.01), resulting in a lower proportion of cows pregnant by 360 dpp (66% vs 78%). Furthermore, the number of services per pregnancy was higher for cows with clinical endometritis than for cows without clinical endometritis (4.4 vs 3.1; p=0.04). Cows with clinical endometritis were 1.6 times as likely to be culled as cows with no signs of clinical endometritis. In conclusion, the prevalence and the impact of clinical endometritis in a pasture-based, extensive dairy production system in Argentina were similar to previously published data from dairy farms with confinement production systems.

Subclinical endometritis and its impact on reproductive performance in grazing dairy cattle in Argentina.

Recently several studies have reported that subclinical endometritis impairs reproductive performance in cattle. Most of the studies were conducted in western industrialized countries under intensive housing conditions. The objective of the present study was to determine the prevalence of subclinical endometritis and its impact on reproductive performance outcomes in clinically healthy postpartum dairy cows in a pasture-based extensive dairy farming system in Argentina. Lactating Holstein cows (n=201) at 18-38 days postpartum (dpp) from three commercial dairy farms in Buenos Aires Province, Argentina, were examined for signs of clinical endometritis by external inspection and manual vaginal examination. Only cows without signs of clinical endometritis i.e. no vaginal discharge were enrolled in this study and examined for subclinical endometritis using the cytobrush technique. Cows with ≥5% polymorphonuclear cells (PMN) in the cytological sample were regarded as affected by subclinical endometritis. All cows were reexamined 14 days later following the same examination protocol. Prevalence of subclinical endometritis 18-38dpp was 38% and decreased to 19% at reexamination. The proportion of cows pregnant at first service was 29% and proportion of cows pregnant at 360pp was 73% and 75% in cows with subclinical endometritis and those without, respectively. The probability of conception at first service, hazards of insemination and pregnancy, respectively, were not affected by subclinical endometritis. Primiparous cows had a greater chance for insemination (HR=0.66; 95% CI=0.47-0.92) and pregnancy (HR=0.63; 95% CI=0.45-0.90) than multiparous cows. In conclusion subclinical endometritis did not affect reproductive performance outcomes in a pasture-based, extensive dairy farming system in Argentina.

A clinical approach to determine false positive findings of clinical endometritis by vaginoscopy by the use of uterine bacteriology and cytology in dairy cows.

Clinical endometritis in dairy cows is defined as mucopurulent or purulent vulvar discharge 21 days or more after parturition. The diagnosis of clinical endometritis is commonly based on vaginal examination. Techniques to reduce the proportions of false negative findings have been described. This paper discusses a clinical approach to determine the proportion of false positive findings that might occur by vaginal inspection. The consequences of false positive findings in dairy practice are unnecessary or inadequate treatments. In research, incorrect diagnoses have an impact on the interpretation of studies on the diagnosis and treatment of clinical endometritis. The objective of the present study was to compare intrauterine bacteriology and endometrial cytology in cows diagnosed with clinical endometritis with findings obtained by vaginoscopy. Clinical endometritis was defined as mucopurulent or purulent vulvar discharge. On two commercial dairy farms, cows were examined 21 to 28 d postpartum. Uterine samples (n = 230) were collected from cows with clinical endometritis with the cytobrush technique to determine the proportion of polymorphonuclear neutrophils (PMN) and to culture smears for aerobic bacteria. Two threshold values for the proportion of PMN (5 and 18%) were chosen as possible indicators for an inflamed endometrium. Common uterine pathogens A. pyogenes and E. coli were found in 33.5 and 10.4% of the samples, respectively. With increasing vaginal discharge score, proportion of samples positive for A. pyogenes increased significantly. The proportion of cows exceeding the thresholds for PMN increased with vaginal discharge score and the presence of A. pyogenes. Considering only the presence of aerobic uterine pathogens and a proportion of PMN above the threshold values of 5 and 18% as indicative for endometritis, a proportion of 17.3 and 28.5%, respectively, of diagnoses by vaginoscopy were false positive.

Viral co-infections in human papillomavirus-associated anogenital lesions according to the serostatus for the human immunodeficiency virus.

In HIV-infected men, human papillomavirus (HPV) infection is strongly linked with the development of anogenital lesions but is not a sufficient factor to explain the neoplastic transformation of such lesions. We investigated the association between HPV and herpesvirus infections in penile and anal lesions from 54 HIV-seronegative and 54 HIV-seropositive men by means of colposcopy, histopathology and in situ hybridization. Our patients showed condyloma acuminata (39%), papular warts (35%) and macular warts (26%). High-grade lesions were predominant in the HIV+ men, whereas low-grade lesions were more frequent in the HIV- men. In the HIV+ group, potential oncogenic HPV were the most frequently detected (83.4%) whereas the "low-risk" HPV were found chiefly in HIV- men (62.1%). The CD4 number was lower in patients showing "high-risk" HPV than in men showing lesions without HPV or with non-oncogenic HPV. HPV types 6/11 were found mainly associated with koilocytosis or with AIN(PIN)I. Oncogenic HPV were more often detected in AIN(PIN)II-III. The herpesviruses DNA detection revealed a higher prevalence of HSVI and -2 than CMV and EBV in the studied biopsies. The frequency of HSV and CMV detection was higher in the HIV+ than in the HIV- men. A link was found between the "high-risk" HPV and the CMV detection whatever the population considered. The detection in HPV lesions of other sexually transmitted viral agents could therefore represent an important means of preventing progression of the anogenital disease, especially in immunosuppressed patients.

Detection of human papillomavirus and human cytomegalovirus in cervical lesions by in situ hybridization using biotinylated probes.

Infections with specific types of human papillomavirus (HPV) have emerged as necessary but not sufficient factors in the development of the majority of cervical cancers. The infection by human cytomegalovirus (HCMV) has also been implicated in both cervical intraepithelial neoplasia (CIN) and cancer. In order to test prevalence of these viral pathogens in genital lesions with suspect cytopathic changes after observation of smears, cervical biopsies from 131 patients were obtained under colposcopic guidance. The biopsies were tested for the presence of HPV and HCMV by the in situ hybridization technique using biotinylated DNA probes on paraffin-embedded sections. Presence of HCMV is twice more frequent in women with HPV-induced cervical lesions (40%) than in women with any detectable HPV (20%). It may be concluded that HCMV might contribute as one synergistic factor in the development of cervical dysplasia.