Nuevo Amanecer (NA‐II), a 10‐week stress management program for rural, low literacy Latina breast cancer survivors.We report results of a community‐based multisite, randomized controlled trial of NA‐II to Spanish‐speaking Latinas with non‐metastatic breast cancer in three rural communities. Women were randomized to receive the program immediately or wait 6 months. Assessments were conducted at baseline, 3 months, and 6 months. Primary outcomes were breast cancer‐specific quality of life domains; secondary outcomes included general distress symptoms and stress management skills. Intention‐to‐treat analyses using repeated‐measures linear regression models estimated changes in slope between groups.Trained peers delivered anxiety at 6 months  as well as three stress management skills: relaxation at 3 months  and 6 months , awareness of tension at 3 months  and 6 months , and coping confidence at 3 months .Of 153 participants , 92% were retained at 6 months. Mean age was 54.8 years (SD = 10.5); 80% had less than high school education. There were no statistically significant treatment × time effects on quality of life. Compared to women in the control group, intervention group women reported greater improvements in Stress management programs delivered by trained peers in rural community settings can reduce anxiety and improve stress management skills among Latina breast cancer survivors. One small study (N = 14) tested a cognitive‐behavioral stress management program in patients with varied types of cancer in rural New England settings.Latina breast cancer survivors report higher rates of anxiety, depression, fear of recurrence, fatigue, pain, and worse health‐related quality of life than their White counterparts.Nuevo Amanecer (NA) program was designed for newly diagnosed Latina breast cancer survivors in urban settings and successfully improved several quality of life domains and decreased breast cancer concerns and depressive and bodily symptoms.NA for Latina breast cancer survivors living in rural settings and expanded the content to include self‐care into survivorship to create NA‐II. In this paper, we report the results of a community‐based multisite, RCT of NA‐II in settings serving rural Latinas with breast cancer.Our original 2NA‐II program and study. Detailed descriptions of study settings, design, recruitment, adaptations, and program content are described elsewhere.We provide a brief description of the 2.1The study was conducted in three California sites serving Spanish‐speaking Latina breast cancer survivors in rural areas that rely on agribusiness. Two were community‐based organizations (CBOs) and one was a community hospital. The sites/community partners were Cancer Resource Center of the Desert (CRCD) , WomenCARE/Entre Nosotras , and Kaweah Delta Health Care District (KDHCD). CRCD is the only non‐profit organization in the Imperial Valley providing Spanish‐language comprehensive cancer support and patient navigation services. WomenCARE is a cancer support program of the Family Service Agency of the Central Coast  and Entre Nosotras is the Spanish‐language arm of WomenCARE. KDHCD is the only Visalia hospital offering comprehensive health care services, including community health workers who provide health education.compañeras at each site), clinicians, survivors, and organizational leaders. Ongoing participation of community members occurred through community consultations, weekly calls and monthly videoconferencing or in‐person meetings with field staff, and review and pretesting of materials by community members. Trained community field staff implemented the study. A lead CBO and partner in the first RCT, Circulo de Vida Cancer Support and Resource Center (CDV), provided clinical supervision of field staff and assisted with training and implementation. CDV is a San Francisco Bay Area Spanish language cancer support services provider.Community‐based participatory research approaches were employed from study conceptualization to dissemination of results. Numerous individuals from each community setting were involved including field staff who were employees of the partner sites  at 3 and 6 months.NA‐II intervention group to a wait‐list control group. The University of California San Francisco Institutional Review Board (IRB) (protocol #16‐18 737) and the KDHCDIRB (protocol # 20160434006) approved the study. UCSF was the IRB of record for the other two sites. Written informed consent was obtained. The study conforms to the U.S. Federal Policy for the Protection of Human Subjects and is registered at http://www.ClinicalTrials.gov (NCT02931552).This was a 6‐month RCT to assess the effectiveness of 2.3Inclusion criteria, which were broad because this was an effectiveness trial, consisted of: (1) Spanish‐speaking Latina (self‐identified ethnicity); (2) diagnosis of Stage 0 to IIIC primary breast cancer; and (3) residing in the selected rural California communities  served by our community partners. Exclusion criteria were terminal illness, metastatic breast cancer diagnosis, or plans to move out of the area in six months.2.4Eligibility screening and recruitment were conducted with a bilingual script by trained bilingual Latinas employed by the community‐based partners. A project director at each site supervised recruiters. For the two CBO sites, recruiters identified potentially eligible women through intake records or outreach and education activities. KDHCD identified potentially eligible women through electronic health records and then mailed them an initial contact letter on their stationery, a study information sheet, and a prepaid refusal postcard to return to KDHCD. If no refusal postcard was received within two weeks, the site recruiter initiated telephone contact with the potential participant. Recruiters verified eligibility through medical or intake records.2.5NA‐II program was adapted from NA to expand its generalizability for rural, low literacy Spanish‐speaking Latina breast cancer survivors throughout survivorship. Adaptation processes for this trial are described in detail elsewhere.NA and NA‐II.NA‐II were based on new formative research with survivors, advocates, patient navigators, medical social workers, and iterative consultations with community representatives from the three rural areas. Results led to expansion of the program from 8 to 10 sessions to accommodate more practice of skills and healthy lifestyles content, greater use of audio‐visuals, further simplification, and creation of a handout for family members providing information on the program.The NA‐II program included ten weekly modules which covered managing the impact of cancer, learning about breast cancer and survivorship, finding cancer information, getting support, identifying helpful and unhelpful thoughts, managing thoughts and mood, stress management techniques, managing activities that affect mood, healthy lifestyles, and goal‐setting. The program emphasized cognitive‐behavioral coping skills training, coaching, and modeling to actively manage stress and emotions. Core components consist of stress management skills training , cognitive reframing (turning unhelpful thoughts into helpful thoughts), effective communication , setting goals, and self‐regulation to achieve goals. Women were provided with general information on breast cancer survivorship  and healthy lifestyles .The compañeras (interventionists) who were Spanish‐speaking breast cancer survivors and at least three years post‐diagnosis with no active recurrence were identified by the community sites. The community Co‐PI, a clinical psychologist at CDV, interviewed candidates using a structured protocol, selecting women who demonstrated excellent interpersonal communication skills, compassion, cultural awareness, and having processed their personal breast cancer experience.Potential Compañeras participated in a 3‐day training (three eight‐hour consecutive days) conducted in Spanish by the PI, Co‐PI, and the Co‐PI's clinical director. Interactive didactic sessions covered psychosocial reactions to breast cancer among Latinas, the theoretical basis of the program, and hands‐on review of the modules with extensive demonstration and role modeling of cognitive‐behavioral stress management skills. Compañeras were trained to model skills for participants.Compañeras and participants were provided with a step‐by‐step Spanish‐language manual covering 10‐weekly sessions. Each week at participants’ homes, one 90‐minute module was presented in‐person using visuals and hands‐on exercises to teach and reinforce concepts and skills.2.6Baseline, three‐month, and six‐month assessments corresponded with our aims of evaluating the 10‐week intervention soon after completion (three months) and retention of benefits after program termination (six months). Trained, community‐based recruiters conducted in‐person 60‐minute baseline assessments (in Spanish) in the participant's home or community partner office. Three‐ and six‐month assessments (30‐minute telephone surveys) were conducted by an experienced, bilingual research associate blinded to participants’ group assignment. Study data were managed using REDCap.2.6.1Primary outcomes were breast cancer‐specific quality of life measures. Secondary outcomes consisted of two types of measures: general distress symptoms and stress management skills. We describe the psychometric properties and descriptive statistics of measures in this sample Table .Breast cancer‐specific quality of life was assessed using the Functional Assessment of Cancer Therapy ‐ Breast (FACT‐B),NA study based on results of psychometric analyses.functional well‐being scale as “enjoyment of life” because items pertain to this more general aspect of well‐being .physical well‐being, 0 to 24; social/family well‐being, 0 to 20; emotional well‐being, 0 to 20; enjoyment of life, 0 to 16; and breast cancer concerns, 0 to 28. The total FACT‐B score  was the sum of the five subscales (range 0‐108). Item‐scale correlations ranged from 0.35 to 0.77 and Cronbach alphas were ≥ 0.74 for all scales, except for breast cancer concerns, which had item‐scale correlations of 0.14 to 0.60 and Cronbach alpha = 0.62 .Anxiety and somatization were assessed with two scales from the Brief Symptom Inventory (BSI).anxiety, 0.62 to 0.75; somatization, 0.35 to 0.63. Cronbach alphas were: anxiety, 0.88; somatization, 0.70.relaxation, awareness of tension, assertiveness, and coping confidence. These skill areas were addressed in the intervention. Women are asked the extent to which they can do each technique when under stress . We used a Spanish translation from a study of Spanish‐speaking prostate cancer survivors.Stress management skills were assessed with the Measure of Current Status Part A (MOCS‐A).relaxation scale assessed women's ability to use relaxation techniques  to reduce tension. The 3‐item awareness of tension scale measures their ability to recognize bodily tension, stressful situations, and when they are becoming tense. The 3‐item assertiveness scale measures the extent to which women are able to ask for help/support when needed and can clearly express their needs. The 5‐item coping confidence scale measures their ability to re‐examine their thoughts to gain a new perspective, decide how to cope with problems, come up with emotionally balanced thoughts, and choose the best coping responses. Item‐scale correlations were: relaxation, 0.49; awareness of tension, 0.58 to 0.63; assertiveness, 0.69 to 0.81; coping confidence, 0.73 to 0.83. Cronbach alphas were: relaxation, 0.66; awareness of tension, 0.76; assertiveness, 0.87; coping confidence, 0.91.The 2‐item Descriptive characteristics included age, language acculturation,2.6.2compañeras completed a structured program tracking form after each session that included the session date, location, and duration. Program adherence was defined as having completed at least 7 of 10 sessions.To assess participant adherence, compañera, at least one intervention session was selected based on convenience, for direct observation for fidelity assessment by the Co‐PI. Of the 7 compañeras, 2 were observed twice. Long travel distances (up to 10 hours) and difficulty coordinating participant, compañera and Co‐PI schedules prevented observation of more visits. Using structured rating scales, the Co‐PI rated interventionists’ compliance with six dimensions on a scale from 1 = not at all to 4 = constantly: extent to which they followed the manual content for that session; explained concepts in easy‐to‐understand language; checked with participant to ensure comprehension; used caring/supportive communication; modeled skills for participants; and provided feedback to participants on skills. Sessions were audiotaped, and reviewed and rated also by two research associates, using the same rating scale. Scores on the six dimensions were calculated as the mean score across coders for each session.For each 2.6.3NA‐II intervention or a wait‐list control group. After the baseline assessment, the recruiter gave the participant a sealed envelope with the next sequential identification number from her stratum indicating to which experimental group she had been assigned. Control group participants received usual care until after the 6‐month assessment at which time they were offered the intervention.Randomization was stratified by recruitment site. Before initiating recruitment, stratum‐specific sequential identification numbers were generated and randomly preassigned in blocks of random sizes. The individual was the unit of randomization with 1:1 allocation to experimental groups. Women were randomized to the 2.6.4d = 0.45as estimated by simulation.A power analysis conducted to establish our enrollment target of 140 women assumed 80% power, two‐tailed α = 0.05, and 90% retention. Intention‐to‐treat analyses focused on treatment × time interaction effects at baseline, three and 6 months, for the primary outcomes of FACT‐B subscale scores. The study was powered for a minimal detectable effect of the proposed design of breast cancer‐specific quality of life scales and secondary outcomes of general distress symptoms , and stress management skills.Likelihood‐based model estimation assumed outcome responses were missing at random.3The RCT was conducted between September 2016 and October 2018. Figure The overall sample had a mean age of 54.8 years  .We found no significant baseline differences between intervention and control groups on demographics, clinical characteristics, quality of life, or distress outcomes, except for educational attainment and self‐rated health, with the intervention group tending to be slightly better educated and more likely to report poor or fair health.anxiety, somatization, depressive symptoms, and perceived stress were low, indicating fairly low levels of general distress symptoms. Breast cancer self‐management skills were fair . There were no significant differences between intervention and control groups at baseline on quality of life, general distress, or stress management skills.Baseline levels of breast cancer‐specific quality of life were low, indicating poor quality of life Table . In geneA large percentage (86%) of the intervention group completed at least 7 of 10 weekly sessions . Six‐month retention was 88% for the intervention group and 95% for the control group . Mean fidelity ratings were: 4.5, extent followed manual; 4.4, used easy‐to‐understand language; 3.9, checked comprehension; 4.4, used caring/supportive communication; 3.8, modeled skills; and 3.9, provided feedback.Primary outcomes: breast cancer‐specific quality of life. There were no statistically significant treatment × time interaction effects on quality of life outcomes at 3 or 6 months (Table 3).Secondary outcomes: general distress symptoms. From baseline to 6 months (3 months post‐intervention), we found significant treatment × time interaction effects on anxiety (P = .049), with the intervention group reporting greater improvement than the control group  . However, there were no significant treatment × time interaction effects for somatization. There were no significant treatment × time effects on depressive symptoms or perceived stress.Secondary outcomes: stress management skills (range 0‐4). The effects of the intervention on stress management skills varied. For relaxation, we observed significant treatment × time interaction effects at 3 months (P < 0.0001) and 6 months (P < 0.001). At 3 months, women in the intervention group experienced greater improvement in their relaxation skills than women from the control group (+0.98 vs −0.07). Positive effects on relaxation skills in the intervention group were maintained (+0.82) at 6 months  and 6 months (P < 0.05). At 3 months, intervention group women experienced greater improvement in awareness of tension than the control group (+0.31 vs −0.19). Effects on awareness of tension in the intervention group were maintained (+0.29) at 6 months  than control group (2.33) (P < 0.05).For coping confidence, we observed significant treatment × time interaction effects at 3 months (P < 0.01). The intervention group improved their coping confidence from 2.45 to 2.57 (+0.12) at 3 months while the control group's scores declined from 2.53 to 2.30 (−0.23) . Quality of life measures were breast cancer‐specific and may not have been as relevant in this study as more general measures of quality of life and distress.Women in this study had several vulnerabilities including rural residence, low socioeconomic status, financial hardship, dependency on public insurance, high level of unemployment and disability, limited English proficiency, and immigrant status. For breast cancer survivors facing these vulnerabilities, providing a sense of confidence in their ability to cope with problems and reduce anxiety may be especially helpful. Since we did not see significant improvements in a general stress measure among the intervention group compared to the control group, it is possible that a more intensive or longer stress management program or a different type of program may be needed to effectively reduce stress in this especially vulnerable group. At a minimum, community partners increased their capacity to deliver evidence‐based stress management skills training.4.1Study limitations include that the sample consisted of Latina breast cancer survivors of Mexican origin only, living in rural California communities. However, many of the cultural values incorporated into the intervention, for example, familismo, spirituality, fatalism and respect are shared by Latinos broadly and cut across national origin. Careful attention was paid in the formative work for this and the prior RCT to ensure that the program met the needs of both rural and urban Latinas. Therefore, it is likely that the program would generalize to Latinas from other national origin groups in the U.S.4.2Among breast cancer survivors, stress has been linked to immunological suppression and cancer progression, thus, reducing stress has important implications for preventing recurrence.5A 2017 report by the National Academies of Sciences, Engineering, and Medicine provides a strong rationale for the community as the locus for confronting health inequities. They argue persuasively that solutions for health disparities involve local community action with a range of partners.The authors declare no conflicts of interest.The contents and views in this manuscript are those of the authors and should not be construed to represent the views of the National Institutes of Health.


The aim of the study was to evaluate oxidant/antioxidant status in 21 Holstein dairy cows with metritis compared to 8 healthy controls. Blood samples were taken during the first 21 days postpartum. Malondialdehyde (MDA), a marker of oxidative stress, total antioxidant status (TAS) and antioxidant parameters such as glutathione peroxidase (GPx), selenium (Se), vitamins A and E and beta-carotene were determined from all cows. The differences in beta-hydroxybutyrate (BHB), non-esterified fatty acids (NEFA), calcium, bilirubin concentrations and aspartate aminotransferase (AST) activity were also monitored, as were milk production and milk composition. Metritis was defined by an unpleasant discharge of varying color (milky-grey/brown/sanguineous) and consistency (muco-purulent/purulent/watery) and by the presence of increased temperature (> 38.5 °C) in cows within 21 days postpartum. Rectal examination revealed increased uterine size, thickened uterine wall and increased uterine tone. The affected cows had significantly reduced daily milk production. Additionally, hematological parameters and haptoglobin concentration were also measured in metritic cows.P <  0.001) was recorded in cows with metritis, while vitamin A and vitamin E concentrations were lower (P <  0.01) compared to healthy cows. Higher BHB (P <  0.05), NEFA (P <  0.05), AST (P <  0.05) and bilirubin (P <  0.001) concentrations was recorded in cows with metritis as compared to the control group. Significant differences in beta-carotene concentration, GPx activity, and Se, TAS and Ca concentrations in cows with metritis compared to control group were not observed in the present study (P >  0.05). Milk production was decreased in the cows with metritis (P <  0.001) and alterations in milk composition were also observed in metritic cows as compared to healthy cows.Higher MDA concentration (The results of the study showed that cows with metritis in early postpartum are exposed to a higher degree of oxidative stress and that the incidence of metritis can negatively affect milk production in dairy cows. The periparturient period is considered critical for high yielding cows (more than 25 l/day), particularly when it comes to metabolism, immunity, health, fertility, milk production and hormonal changes , 2. The 2 alpha or an intrauterine antimicrobial is effective [Escherichia coli, Trueperella pyogenes, Fusobacterium necrophorum, Prevotella melaninogenica, Streptococcus pyogenes, Bacteroides spp., Pseudomonas spp. and Staphylococcus spp. are common pathogens in endometritis and metritis [The lumen of the uterus after calving is often contaminated with bacteria that may cause disease that can also lead to infertility. The immune response of postpartum cows and the number and type of bacteria are important factors in the development of uterine disease . Polymorffective . When thffective , 13. On ffective . Escherimetritis , 15. Up metritis , 16.Clinical signs of metritis are well known. Infection usually occurs within the first 21 days (most often 10 days) after calving. Malodorous discharge, from reddish brown watery discharge to more viscous lighter discharge, from the uterus is an essential clinical sign of metritis. Fever > 39.5 °C) is a frequent accompanying symptom. Other clinical signs such as apathy, decreased milk yield, dehydration, anorexia, increased heart rate and dehydration can be also observed in the metritis. An enlarged uterus can be detected by rectal examination. Metritis often occurs in association with a retained placenta, the birth of twins, dystocia and premature birth .5 °C is .Oxidative stress in the periparturient period is known to be one of the factors contributing to increased susceptibility to diseases like mastitis, metritis, mammary edema and retained fetal membranes , 18. ManIn relation to current literature, we have attempted to provide a comprehensive overview / relationship in the diagnosis of metritis by clinical and laboratory examination, oxidative stress using oxidative and antioxidant markers, the basic metabolic profile characteristic of postpartum period in cows and milk production and its composition during the first 30 days of lactation. The main purpose of the study was to monitor the relationship between oxidative stress and the occurrence of metritis postpartum in dairy cows. Therefore, the objective of this study was to compare the marker of oxidative stress and antioxidant status – malondialdehyde (MDA), total antioxidant status (TAS), glutathione peroxidase (GPx), selenium (Se), vitamins A and E, and beta carotene, and additionally the metabolic profile – beta-hydroxybutyrate (BHB), non-esterified fatty acids (NEFA), calcium (Ca), aspartate aminotransferase (AST) and bilirubin between cows with metritis and a healthy control group. Based on the above findings, we expect an increase in oxidative stress indicators and a decrease in antioxidants in metritic cows with compared to the control group. Another objective was to assess whether metritis affects the milk production of dairy cows. Milk production was recorded and also milk composition including milk fat, protein, lactose, urea and somatic cell count (SCC) were determined in cows of both groups.The study was carried out using 21 Holstein dairy cows with metritis (M group) and 8 healthy controls (CO group), with a mean milk production of 10,249 l, at a farm located in the village of Uherčice . The farm was chosen because of the incidence of metritis in the cows and also because of the good cooperation with the staff. The experiment was carried out over 5 months (april-august 2018). Cows in the control group had no complications of the diseases, none were treated for diseases during the experimental period, and none of them had calving complications (only single pregnancy). Cows in M group were selected on the basis of clinical signs of disease (listed below). Cows (ranging) from the first to the sixth lactation were included in the experiment, specifically the parity of CO group was 1–4 (average 1.75) and M group 1–6 (average 2.29). There were no instances of death or culling. All of the cows were fed a total mixed ration (TMR) according to the antepartum and the postpartum period . Metritis was defined by an unpleasant discharge of varying color (milky-grey/brown/sanguineous) and consistency (muco-purulent/purulent/watery) and then by the presence of increased temperature (> 38.5 °C). Rectal temperature and discharge character, color and odor were observed  without anticoagulant for serum determination  and into Hemos sampling tubes  with heparin anticoagulant for determination of whole blood  and plasma (MDA). Blood samples for determination of hematological parameters  were only collected from metritic cows into sampling tubes with EDTA (ethylenediaminetetraacetic acid) anticoagulant. For serum samples, blood was allowed to clot and kept at room temperature until the separation of serum. Clotted blood was centrifuged at 3000 rpm for 10 min. Serum samples were either immediately used for analysis or stored at − 70 °C. For plasma samples, whole blood was centrifuged at 3000 rpm for 10 min. Plasma samples were stored at − 70 °C until analysis. Blood samples for hematological determination were analyzed immediately after collection.Hematological parameters such as WBC, lymphocytes, monocytes, granulocytes and RBC were measured using an automatic BC–2800 Vet hematology analyzer . Haptoglobin concentration was analyzed with a Haptoglobin kit  using a colorimetric assay by an automatic Konelab 20XT biochemical analyzer .3 and H2O2 using the microwave digestion system ETHOS TOUCH CONTROL  followed by evaporation. Whole blood GPx activity was measured with a RANSEL kit  using a UV method based on that of Paglia and Valentine [The plasma MDA concentration was determined using the high-performance liquid chromatography (HPLC) system Ultimate 3000 , after prior derivatization of MDA with 2, 4-dinitrophenylhydrazine as described previously . The selalentine . Serum TSerum NEFA and BHB were measured using standardized kits supplied by Randox Laboratories Ltd. The other biochemical parameters such as bilirubin and AST in serum were determined using commercial kits . An automatic Konelab 20XT biochemical analyzer  was used for determination. Serum Ca concentration was analyzed using flame atomic absorption spectrometry - F AAS . Blood samples for Ca determination were diluted with EDTA 3.The results obtained were tested for the homogeneity of variances (Hartley-Cochran-Bartlett test) and the normality of distribution (Shapiro-Wilk test). In the event of non-normality, outliers were excluded. The data were analyzed statistically by one-way analysis of variance (ANOVA) followed by the Fisher LSD post-hoc test. All results were expressed as mean value (x) ± standard deviation (SD).9/L), lymphocytes (2.19 ± 0.88 × 109/L), monocytes (0.70 ± 0.20 × 109/L), granulocytes (4.00 ± 1.98 × 109/L) and RBC (5.93 ± 1.13 ×  1012/L) and higher haptoglobin concentration (1.07 ± 0.57 g/L) in cows with metritis compared to reference values of these parameters.Mean values of hematological parameters and haptoglobin concentration in cows with metritis are shown in Table P <  0.001), while vitamin A  and vitamin E  concentrations were decreased compared to the control group . In the present study, no significant differences in GPx activity and Se and TAS concentrations were observed in cows with metritis (P > 0.05).As shown in Table P <  0.05), NEFA , AST  and bilirubin  were recorded in cows with metritis, while the Ca concentration was slightly decreased, though this was not significant compared to the healthy cows . The difference in milk production between cows with metritis and healthy control cows is shown in Fig. 3 cells/ml; P > 0.05), whereas protein, lactose and milk urea did not show significant differences between the metritic group and the control group (P > 0.05).Milk production and values of milk fat, protein, lactose, SCC and milk urea concentration in cows with metritis and in healthy cows in the first month of lactation are shown in Table The detection of metritis in cows was based on clinical and rectal examination. Rectal examination revealed increased uterine size, thickened uterine wall and increased uterine tone. The main factor for the diagnosis of metritis was the evaluation of discharge of varying color and consistency. Another parameter used to diagnose the disease was an increased rectal temperature. In our study, the rectal temperature in metritic cows ranged from 38.4–39.6 °C Table . Thus, a9/l) was also observed in metritic cows compared to physiological range in cattle [9/l) and monocytes (0.70 ± 0.20 cells × 109/l) was decreased in dairy cows with metritis. The results are in agreement with the previous study by Barragan et al. [6/ml), lymphocytes (4.13 ± 0.58 cells × 106/ml) and monocytes (0.10 ± 0.20 cells × 106/ml) were recorded in cows with clinical metritis. The reason for decreased WBC in dairy cows with metritis is apparently the accumulation of leukocytes in the uterine lumen, as already mentioned in previous studies. Barragan et al. [6/μl) compared to cows without clinical metritis as a cause of internal hemorrhage due to rupture of the uterine submucosa blood vessels. The number of erythrocytes was decreased in cows with metritis (5.93 ± 1.13 cells × 1012/l) in our study as compared to physiological ranges in cattle [In addition to clinical signs, the determination of hematological parameters and haptoglobin concentration were also used to verify the occurrence of metritis in dairy cows. The total count of WBC is used as a marker of infections and inflammation. A decrease in WBC may occur in the case of bacterial and viral infections such as metritis, mastitis and pneumonia. Leukocytes, particularly neutrophils, are transferred to the damaged tissue or the site of infections within 2 h . In the n cattle , 29  in cows with metritis, while a decrease in vitamin concentrations (P < 0.01) was also observed. The results of the study by Kizil et al. [In the present study, the plasma MDA concentration was increased . This could be explained by the fact that although the concentration of vitamins was reduced in metritic cows, other parameters with an antioxidant effect, such as for example the bilirubin concentration, may be increased . However, additional information about feed intake in dairy cows would be required to suggest a hypothesis.Furthermore, metabolic profiles including NEFA, BHB, Ca, bilirubin concentration and AST activity have been used in an attempt to monitor metritis in dairy cows more closely. Insufficient energy intake in the periparturient period in dairy cows results in an increase in NEFA concentrations as a response to lipomobilization . Lower fIngvartsen and Moyes  and MartP < 0.05) and bilirubin concentration (P < 0.001) were reported in cows with metritis compared to healthy cows. The study by Moretti et al. [The increase in serum AST concentrations after calving may be associated with increasing synthetic activity in the liver due to protein mobilization  or with i et al.  found thP < 0.05) are associated with milk production in individual groups. Milk fat content is known to decrease with increasing milk yield and in the first half of lactation. The lower fat content of milk in the control group may be due to the high milk production in these cows . On the other hand, increased fat content of milk in early lactation is associated with the development of energy deficiency and subsequent occurrence of ketosis and lipomobilization syndrome. Lipolysis occurs during NEB and fatty acids used for the synthesis of milk fat are released from the fat reserves of dairy cows [3 cells/ml; P = 0.05).Metritis and uterine diseases in general are associated with decreased milk yield , 38, 63.iry cows , which mIn cows with post-partum metritis, an elevated marker of oxidative stress and conversely a decline in antioxidant concentrations were observed compared to healthy cows. The metabolic profile, including energy balance indicators and liver markers, also demonstrated significant differences between the groups. The results of the study showed a relation between oxidative stress and metritis. This suggests that cows with metritis in early postpartum are exposed to a higher degree of oxidative processes. Our findings also showed that the incidence of metritis negatively affected milk production and composition in dairy cows. It can be concluded that monitoring the energy balance , the total mixed ration composition and metabolic imbalances followed by possible preventive action in the prepartal period is important and can contribute to prevent the incidence of such diseases and possible negative consequences in dairy cows. For further research, it would be advisable to follow the developmental line from the prepartal to the postpartum period in cows with metritis compared to a control group.


Leptospira is widespread in rodents, the most studied reservoir and the main hosts involved in its transmission. In Italy, among rodents, Hystrix cristata (crested porcupine) is the largest species and it is distributed all over the country. In this paper, the isolation and characterization of pathogenic Leptospira spp. from the kidney of H. cristata is reported for the first time. During Autumn 2018, Leptospira detection by real‐time PCR and isolation were performed from kidneys of two died female porcupines (an adult and a porcupette). Only for porcupette kidney sample, real‐time PCR for pathogenic Leptospira tested positive. The isolated strain was identified as Leptospira interrogans serogroup Pomona serovar Pomona, using the three schemes of multilocus sequence typing. The results show that H. cristata could be a Leptospira host. The infection of serovars Pomona could be related to the habitat shared with wild boar, a typical reservoir host for this serovar.Pathogenic  Leptospira spp. from the kidney of a crested porcupine (Hystrix cristata) is reported for the first time. The isolated strain was identified as Leptospira interrogans serogroup Pomona serovar Pomona.In this paper, the isolation and characterization of pathogenic  The first one was recovered in the area of Pescia Fiorentina , whereas the second one in the area of Arcille (Campagnatico). At the clinic, animals were treated with enrofloxacin and dexamethasone for about 3 days before the death. From each carcass, during necropsy, kidneys and a blood sample from the heart cavity were collected. Before the necropsy, both animals were sexed and weighted and the age class was estimated , Canicola , Pomona , Grippotyphosa , Tarassovi , Bratislava , Hardjo , Castellonis , Copenhageni , Bataviae , Australis , Zanoni , Saxkoebing , Sejroe , Poi , Mini , Lora , Hardjo , Autumnalis  and Hebdomadis .Blood samples were centrifugated at 1,200 MAT OIE, . Titre o2.33 from each porcupine kidney was homogenized with 5 ml of sterile water. One ml of homogenate was cultured in 5 ml of EMJH, incubated at 30 ± 1°C for 120 days and checked every 10 days under dark‐filed microscopy to assess bacterial growth.The kidney samples were cultured in Ellinghausen–McCullough–Johnson–Harris (EMJH) medium (Difco). A portion of 10 cmIn case of positive cultures, subcultures were performed to maintain the isolated strains alive.2.4Leptospira were genotyped using a multilocus sequence typing scheme encompassing housekeeping genes . Amplicons were further sequenced (BMR Genomics) using the same primer sets and analysed using BioEdit Software Hall, .2.5DNA was extracted from each kidney using the Quick‐DNA Plus Kits (Zymo Research) according to the manufacturer's instructions.lipL32 gene Taqman RealTime PCR was performed on a Rotorgene Corbett 6000 (Corbett Research) to detect pathogenic leptospires  prior to incubation. A polyclonal horse serum (1 drop diluted in 1 ml of PBS) was used as a secondary antibody for 30 min at room temperature (Vector Laboratories). Antibody binding was detected using a streptavidin‐biotin‐peroxidase kit (Vector Laboratories). The enzymatic reaction was developed with the use of 3‐1‐diaminobenzydine  as a substrate. Stained slides were subsequently counter‐stained in haematoxylin for 40 s followed by a wash in tap water, dehydration in graded alcohols , and clearance with xylene. Sections were mounted in DPX . As a positive control, a pig kidney culture‐positive for serogroup Pomona and a mouse kidney culture‐positive for serogroup Grippotyphosa were used.Representative portions of the kidneys collected during necropsy were routinely processed, paraffin‐embedded and 5‐µm‐thick sections were stained with haematoxylin and eosin, Masson trichrome Goldner and Warthin Starry stains. Tissue sections were also submitted to immunohistochemistry. Antigen retrieval was achieved on the slides by placing them in a bath of 10 mmol/L citric acid (pH 6) and boiling for 16 min in an 800‐W microwave oven. The slides were dried at room temperature and washed with running tap water. A peroxidase block was performed, and the slides were incubated with specific rabbit antisera against 3Leptospira serogroup Pomona an intense immunoreactivity for leptospiral antigen was detectable within the tubular epithelia cells and in cellular debris in tubular lumen  and a porcupette (around 2 months old) of 1.4 kg. The kidney of the porcupette showed small gray‐white focal lesions, mainly located in the renal cortex and varying from 1 to 2 mm in diameter. Microscopically, a mild chronic interstitial nephritis was present, characterized by vacuolar degeneration of the tubular epithelium and scattered interstitial foci consisting of lymphocytes and plasma cells  140 for scheme 1, ST 4 for scheme 2 and ST 58 for scheme 3.Both sera samples resulted negative to MAT for all 4Giardia duodenalis , as well  of porcupine sera resulted positive to anti‐Leptospira antibodies detection; Icterohaemorrhagiae resulted the most prevalent serogroup (4 positive sera), followed by serogroup Pomona and Australis . Titres of 1:400 and 1:100 were recorded for serogroup Pomona  which are present in Tuscany  with an high‐density population (Massei et al., Leptospira serovars, such as Pomona, in the environment (Bertelloni et al., Rodents are well known important Leptospira from crested porcupine suggests that H. cristata could be a new potential natural host for this bacterium.The isolation of Leptospira serovar Pomona infection in crested porcupine. Despite the single positive sample, this result could be a useful contribute to the description of the epidemiology of leptospirosis. Further investigations are needed to specifically determine the role of crested porcupine as accidental or as maintenance host for Leptospira.Authors are aware of the limit of the study, since it represents a case report of 5reservoir of Leptospira. The results obtained in this paper show that H. cristata could be a Leptospira host, as hypothesized for other hystricomorph rodents. The infection by serovars Pomona, typically observed in swine and wild boar, could suggest an adaptability and/or a change in host range for this serovar, as assumed for other serovars. Further investigations are needed to clarify the role of this peculiar rodent in the epidemiology of leptospirosis in Italy.Leptospirosis is one of the most widespread and emerging zoonotic disease in the world, and wild animals were known to be All authors declare no conflict of interests.Giovanni Cilia: Data curation; Formal analysis; Investigation; Methodology; Writing‐original draft; Writing‐review & editing. Fabrizio Bertelloni: Data curation; Formal analysis; Methodology; Writing‐original draft; Writing‐review & editing. FRANCESCA COPPOLA: Data curation; Investigation; Writing‐original draft; Writing‐review & editing. Barbara Turchi: Data curation; Investigation; Methodology; Writing‐review & editing. Claudia Biliotti: Data curation; Investigation; Writing‐review & editing. Alessandro Poli: Data curation; Investigation; Methodology; Writing‐review & editing. Francesca Parisi: Data curation; Investigation; Methodology; Writing‐review & editing. Antonio Felicioli: Conceptualization; Data curation; Supervision; Writing‐original draft; Writing‐review & editing. Domenico Cerri: Conceptualization; Data curation; Resources; Supervision; Writing‐review & editing. Filippo Fratini: Conceptualization; Data curation; Investigation; Methodology; Resources; Supervision; Writing‐original draft; Writing‐review & editing.The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethical approval was required.


Inferential statistics and Pearson correlations were used to investigate relationships between body‐mass index, type 2 diabetes, hypertension and proteinuria. Mean ± SD BMI was 28.6 ± 5.8 kg/m2 (n = 374). Overweight and obesity class 1 were more common in males (P = .02). Amongst n = 123 individuals with obesity and chronic kidney disease, mean ± SD age, n (%) female and median[IQR] eGFR were 64.1 ± 14.2 years, 52 (42.3%) and 29.0[20.5] mL/min/BSA, respectively. A positive correlation between increasing body‐mass index and proteinuria was observed in such patients , which was stronger in males and those with CKD stages 4 and 5. Mean body‐mass index was 2.3 kg/m2 higher in those treated with 4‐5 versus 0‐1 antihypertensives (P = .03). Amongst n = 59 patients with obesity, chronic kidney disease and type 2 diabetes, 2 (3.5%) and 0 (0%) were prescribed a GLP‐1 receptor analogue and SGLT2‐inhibitor, respectively. Our data provides a strong rationale not only for measuring body‐mass index but also for acting on the information in nephrology practice, although prospective studies are required to guide treatment decisions in people with obesity and chronic kidney disease.Obesity is a treatable risk factor for chronic kidney disease progression. We audited the reporting of body‐mass index in nephrology outpatient clinics to establish the characteristics of individuals with obesity in nephrology practice. Body‐mass index, clinical information and biochemical measures were recorded for patients attending clinics between 3 Obesity is recognized to exacerbate proteinuria amongst people with chronic kidney disease. The magnitude of this relationship, and how it is influenced by gender and chronic kidney disease stage, is inadequately described. In addition, whether or not a continuous relationship exists between increasing BMI and proteinuria amongst people with obesity and chronic kidney disease is unknown.Data on the incorporation of GLP‐1 receptor analogues and SGLT2‐inhibitors, which have metabolic, weight and renal benefits in people with type 2 diabetes, into real‐world clinical practice for the management of high‐risk patients with chronic kidney disease, are lacking.Outside of NHANES surveys in the United States, reports on the prevalence of obesity and associated comorbidity burden amongst people with chronic kidney disease are sparse. In addition, insights from such data are limited by the lack of granular information on physician‐assigned diagnoses of chronic kidney disease aetiology.Obesity is over‐represented in people with chronic kidney disease compared with background rates in the general population. Over 60% of the chronic kidney disease burden amongst people with obesity is attributable to diabetic kidney disease and hypertensive nephropathy; conversely, alternative chronic kidney disease aetiologies, including primary glomerular diseases, obstructive nephropathy and interstitial renal diseases, remain common in people with obesity and account for over 35% of chronic kidney disease cases in this setting.Amongst people with obesity and chronic kidney disease, increasing BMI associates with greater proteinuria and antihypertensive usage. The relationship between increasing BMI and proteinuria is stronger in males and those with advanced chronic kidney disease (stages 4 and 5). As a means of reducing the severity of hypertension and proteinuria, and consequently accelerated chronic kidney disease progression, intentional weight loss strategies should be explored in these particularly high‐risk subgroups of people with obesity and chronic kidney disease.The majority of people with obesity, chronic kidney disease and type 2 diabetes in our cohort were treated with weight‐promoting diabetes therapy (insulin and sulphonylureas), rather than newer therapies with weight‐lowering effects as well as established end‐organ benefits in the kidney . In our study cohort reflective of real‐world contemporary nephrology practice, obesity was infrequently addressed as a modifiable risk factor for chronic kidney disease progression.1Obesity, hypertension and type 2 diabetes mellitus (T2DM) constitute inter‐related pandemics that have increased the prevalence of chronic kidney disease (CKD).Reducing the severity of obesity with metabolic surgery decreases the incidence of albuminuria and end‐stage kidney disease (ESKD) over long‐term follow‐up.22.1Permission was obtained from St Vincent's Healthcare Group, Dublin, Ireland for a prospective clinical audit (reference number 2018/2244) evaluating characteristics of people with obesity attending nephrology outpatient clinics. Under clinical audit guidelines, informed consent from individual participants was not obtained, but all data were handled as per the General Data Protection Regulation guidelines (EU), 2016/679. All procedures performed were in accordance with the ethical standards of the institutional audit committee and with the 1964 Helsinki declaration and its later amendments. Amongst people with obesity and CKD, cross‐sectional relationships between BMI, Chronic Kidney Disease‐Epidemiology Collaboration (CKD‐EPI) estimated glomerular filtration rate (eGFR), urine protein‐to‐creatinine ratio (uPCR), and usage of antihypertensives and glucose‐lowering medications are also reported. People with ESKD on haemodialysis or with a prior kidney transplant were excluded. Pregnant women, women in the post‐partum period for less than 3 months and individuals who self‐reported anabolic steroid use or with missing clinical and laboratory data were excluded.2.2rd August, 2018 and 18th January, 2019 using a Seca 701 electronic scale and Seca 220 stadiometer, respectively. Body‐mass index (BMI) (kg/m2) was calculated: body weight (kg)/(height × height [m2]).Body height and weight were routinely measured in nephrology outpatient clinics between 32.31c]) were analysed via routine clinical biochemistry services at St Vincent's University Hospital, Dublin. Haemoglobin was measured and reported by the Haematology laboratory. The Pathology Department is accredited by the Irish National Accreditation Board to ISO 15189:2012 standards. All analyses, with the exception of haemoglobin and HbA1c, were performed on the Roche/Hitachi Cobas 8000 analyser series; c702 and e602 . The enzymatic creatinine method was used, traceable to serum reference manual 914 and isotope‐dilution mass spectrometry. HbA1c was measured by high‐performance liquid chromatography . Haemoglobin was measured on the Sysmex XE‐5000 differential analyser . Urine protein was measured using turbidimetry with benzethonium chloride, traceable to a NIST standard. CKD‐EPI eGFR, calculated using standard formulae, was recorded and expressed as mL/min/body surface area (BSA).All recorded clinical biochemistry tests . Continuous variables with normal and skewed distributions are presented as mean ± SD and median [interquartile range], respectively. One‐way between‐group ANOVAs and Kruskal‐Wallis tests were used to assess for differences across the BMI categories of obesity (three groups) in continuous variables with normal and skewed distributions, respectively. Independent sample t tests and Wilcoxon rank‐sum tests were used to assess for gender differences (two groups) in continuous variables with normal and skewed distributions, respectively. Univariate relationships between BMI, uPCR and HbA1c were investigated by Pearson correlations. The functions “ggscatter” and “ggboxplot” from the R package “ggpubr” were used to generate scatterplots and boxplots, respectively.P <.05 was considered statistically significant.RStudio version 3.6.1 was used for analysis. BMI distribution and prevalence of BMI categories at study enrolment were summarized by descriptive statistics. Categorical variables are presented as frequencies and percentages and were compared between groups using 33.1P = .06) and had a 1.3% higher prevalence of Caucasian ethnicity (P = .07). Mean BMI was 28.6 ± 5.8 kg/m2, and did not significantly differ between males and females (P = .61). Prevalent BMI distributions were: <18.5 kg/m2 , 18.5 to 24.9 kg/m2 , 25 to 29.9 kg/m2 , 30 to 34.9 kg/m2 , 35 to 39.9 kg/m2 , ≥40 kg/m2 . Overall, n = 132 individuals (35.3%) were affected by obesity, compared to a population average in Ireland of 23%.P = .02). Figure BMI was measured in 384 individuals over the 6‐month study period; n = 10 people were excluded for: active haemodialysis treatment (n = 3), missing clinical and laboratory data (n = 3), pregnancy (n = 2), <3 months post‐partum (n = 1), and anabolic steroid use (n = 1). Table 3.22) attended nephrology clinics during the study period. Nine patients did not have confirmed CKD. Of these, four were attending nephrology clinics for blood pressure management, two for evaluation of high serum creatinine, one for evaluation of proteinuria, one for nephrolithiasis and one for recurrent urinary tract infections. Table P = .04). The majority of the cohort had hypertension (87%) and dyslipidaemia (63%), while almost 25% had established coronary artery disease. Over 60% and 50% of people were treated with renin‐angiotensin‐aldosterone system (RAAS) blockade and statins, respectively. Overall, 50 (40.7%) patients had DKD and together, DKD and hypertensive nephropathy accounted for almost 65% of cases of CKD. Conversely, alternative CKD aetiologies remained common in people with obesity and accounted for over 35% of CKD cases. Often, individuals with obesity had advanced CKD; stage 4 was the most common CKD class and median eGFR was 29 [20.5] mL/min/BSA. No significant differences in office blood pressure, metabolic parameters (HbA1c and lipid indices), anaemia parameters (haemoglobin and iron indices), cardiovascular comorbidities, CKD stage and aetiology, degree of proteinuria and medication usage were observed across categorical obesity classes.In total, 132 people with obesity  was observed between increasing BMI and increasing uPCR in individuals with obesity. The relationship between BMI and uPCR was stronger in males  than females , although the smaller sample of females may have limited statistical power. Increasing BMI also had a greater impact on uPCR in those with CKD stages 4 and 5 . No relationship between BMI and HbA1c was observed amongst all patients with obesity and CKD  and amongst the subgroup of patients with obesity, CKD and type 2 diabetes . Figure 2), BMI was significantly higher in those treated with 4‐5 antihypertensives . Figure P < .001) and median uPCR was 118 mg/mmol higher (P = .02) in those treated with 4‐5 antihypertensives. The size of individual data points in Figure Figure 3.42 were treated with either a GLP1RA or SGLT2i.Table 4This study provides insight into the BMI distribution of males and females attending general nephrology clinics. Obesity (35.3%) was common in outpatient nephrology practice. Obesity class 1 was more common in men, while normal weight and obesity classes 2 and 3 were more frequently observed in females. Amongst individuals with obesity and CKD, a positive correlation was observed between increasing BMI and uPCR, a relationship, which was stronger in males and those with more advanced CKD stages (eGFR <30 mL/min/BSA). BMI was higher in individuals treated with 4‐5 compared with 0‐1 antihypertensives. Individuals with obesity, CKD and T2DM were frequently prescribed RAAS blockade, statins, insulin and sulphonylureas, but infrequently prescribed diabetes therapy with weight‐lowering effects, including GLP1RAs and SGLT2is.Obesity was more common in people with CKD than expected based on trends in the general Irish population. In the 2015 Healthy Ireland survey of 6,142 people aged ≥15 years, the prevalence of obesity was 23%.The prevalence of obesity amongst people with CKD is even higher in the United States, where it increased from 38.1% between 1999 and 2002 to 44.1% between 2011 and 2014.2) and CKD. Obesity‐related glomerular hyperfiltration, which is an independent predictor of adverse cardiovascular outcomes, and the obesity paradox may have contributed to the high burden of cardiovascular disease even amongst individuals with less severe obesity (WHO classes I and II) in our cohort.2 in a single‐centre series from the United States.Amongst people with obesity and CKD, the complications of hypertension, dyslipidaemia and diabetes mellitus were very common and present in over 85%, 60% and 50% of patients, respectively. No major differences in cardiovascular comorbidities or CKD stage were observed across incremental categorical classes of obesity. This may be partly related to the relatively low number of patients (n = 18) with obesity class 3 (BMI ≥ 40 kg/m2 higher in those treated with 4 to 5 compared with 0 to 1 antihypertensives. These findings suggest the presence of a continuous gradient between increasing BMI and increasing severity of proteinuria and hypertension, the two major modifiable determinants of CKD progression,2 a relative contraindication to kidney transplantation.Despite the lack of observed difference in CKD severity across obesity classes 1 to 3 in our cohort, when BMI was treated as a continuous variable, a significant positive correlation between increasing BMI and increasing uPCR was observed amongst people with obesity and CKD. Importantly, this association was stronger in males and those with CKD stages 4 and 5. Mean BMI was also 2.3 kg/mPatients with obesity, T2DM and CKD are an important subgroup to target with intensification of therapy given the progressive nature of DKD. In a longitudinal study of patients with type 2 DKD, annual decline in CKD‐EPI eGFR decreased from −5.6 to −3.1 mL/min/BSA/year after intensification of therapy through an integrated nephrology and diabetology clinic.Nevertheless, median eGFR in our study cohort was 29 mL/min/BSA; SGLT2is are not yet licensed in this setting due to concerns that they may lack glycaemic efficacy in those with eGFR <45 mL/min/BSA.Our data should be interpreted taking account of certain limitations. The cross‐sectional nature of this study hampered attempts to infer causality. The predominance of white Caucasians in our cohort limits the applicability of our findings to adults from other racial and ethnic backgrounds. The extent to which our findings are generalizable requires confirmation in international cohorts where greater ethnic diversity amongst patients with CKD is observed, particularly given the higher risk of CKD and ESKD amongst African Americans.5In conclusion, obesity was common amongst individuals attending nephrology clinics. Class 1 obesity was more common in males; normal weight and obesity classes 2 and 3 were more frequently observed in females. The severity of obesity did not significantly influence the prevalence of cardiovascular complications or CKD stage amongst people with obesity and CKD. Increasing BMI associated with increasing proteinuria amongst people with obesity and CKD, a relationship which was stronger in males and those with CKD stages 4 and 5. Increasing BMI also associated with greater antihypertensive usage in patients with obesity and CKD. The majority of people with obesity, T2DM and CKD were treated with medications that promote weight gain, including sulphonylureas and insulin; GLP1RAs and SGLT2is were infrequently prescribed in this setting. Prospective studies of medical and surgical intentional weight loss strategies in people with obesity and CKD evaluating renal, cardiovascular, and mortality outcomes are warranted.ClR discloses personal fees outside of the submitted work from Novo Nordisk, GI Dynamics, Eli Lilly, Johnson and Johnson, Sanofi, Aventis, Astra Zeneca, Janssen, Bristol‐Myers Squibb, and Boehringer‐Ingelheim. The authors declare that they have no competing interests.The anonymised dataset used during the current study is available from the corresponding author on reasonable request. Statistical code written in the R programming language for the current study is also available from the corresponding author on reasonable request.William P. Martin, Neil G. Docherty, Carel W. le Roux and John Holian designed the study. William P. Martin, Aisling O'Riordan, Alan J. Watson and John Holian provided clinical care to the study cohort. William P. Martin, John Coleman and Ludmilla Dellatorre‐Teixeira collected BMI data for the study cohort. William P. Martin, Jessica Bauer and John Coleman collected clinical and laboratory information for the study cohort, with assistance from Janice L.V. Reeve. Janice L.V. Reeve and Patrick J. Twomey quality assured the laboratory analysis. William P. Martin performed the statistical analyses. William P. Martin, Neil G. Docherty, Aisling O'Riordan, Alan J. Watson, Carel W. le Roux and John Holian interpreted the data. William P. Martin, Neil G. Docherty, Carel W. le Roux and John Holian drafted the manuscript with critical input from all authors. All authors reviewed and approved the final version of the manuscript.


Bemisia tabaci cryptic species are known to contribute to host fitness and environmental adaptation. The genetic diversity and population complexity were investigated for endosymbiont communities of B. tabaci occupying different micro‐environments in Pakistan. Mitotypes of B. tabaci were identified by comparative sequence analysis of the mitochondria cytochrome oxidase I (mtCOI) gene sequence. Whitefly mitotypes belonged to the Asia II‐1, ‐5, and ‐7 mitotypes of the Asia II major clade. The whitefly–endosymbiont communities were characterized based on 16S ribosomal RNA operational taxonomic unit (OTU) assignments, resulting in 43 OTUs. Most of the OTUs occurred in the Asia II‐1 and II‐7 mitotypes , while the Asia II‐5 microbiome was less complex. The microbiome OTU groups were mitotype‐specific, clustering with a basis in phylogeographical distribution and the corresponding ecological niche of their whitefly host, suggesting mitotype‐microbiome co‐adaptation. The primary endosymbiont Portiera was represented by a single, highly homologous OTU (0%–0.67% divergence). Two of six Arsenophonus OTUs were uniquely associated with Asia II‐5 and ‐7, and one occurred exclusively in Asia II‐1, two only in Asia II‐5, and one in both Asia II‐1 and ‐7. Four other secondary endosymbionts, Cardinium, Hemipteriphilus, Rickettsia, and Wolbachia OTUs, were found at ≤29% frequencies. The most prevalent Arsenophonus OTU was found in all three Asia II mitotypes (55% frequency), whereas the same strain of Cardinium and Wolbachia was found in both Asia II‐1 and ‐5, and a single Hemipteriphilus OTU occurred in Asia II‐1 and ‐7. This pattern is indicative of horizontal transfer, suggestive of a proximity between mitotypes sufficient for gene flow at overlapping mitotype ecological niches.Endosymbionts associated with the whitefly  Bemisia tabaci in Pakistan. It was shown for the first time that B. tabaci mitotypes and their associated secondary endosymbiont are geographically codistributed and that the distributions are consistent with climatic gradient niches, suggestive of ecologically associated whitefly–endosymbiont co‐adaptation.This manuscript describes the diversity of endosymbionts associated with  They are not considered to be as vital for survival of B. tabaci, as is the primary endosymbiont Portiera, but instead, serve as source of B vitamins , have been reported at low frequencies , Baumann,  by contrBaumann, . CospeciBaumann, , includiBaumann, , leafhopBaumann, , mealybuBaumann, , and psyBaumann, . HoweverBaumann, , represeB. tabaci can be influenced by endosymbiont community composition, which in turn can alter the distribution of different vector mitotype–virus combinations. In Pakistan and India, studies have shown that B. tabaci mitotypes native to Asia that transmit cotton leaf curl disease‐begomoviruses , can influence the spatio‐temporal dynamics in whitefly mitotype diversity and population structure  sequence as a molecular marker, whereas the composition and frequency of bacterial operational taxonomic units (OTUs) were based on ribosomal 16S RNA gene (rDNA) sequence comparisons. Also, the potential for horizontal transmission of secondary symbionts between the endemic mitotypes of B. tabaci was investigated by PCR amplification, sequencing, and phylogenetic analysis of the 16S rRNA gene sequence.The objectives of this study were to determine the composition and rate of fixation of bacterial primary and secondary endosymbionts in relation to the spatial distribution of 22.1n = 3–9). This may be further explained by the minimal to no use of pesticides in the urban areas, which leaves the natural enemies relatively undisturbed, compared to agricultural areas where pesticide use during the cotton‐growing season is frequent to control the whitefly vector, with the caveat that its frequent use often leads to increases in whitefly population sizes  and II‐7 (n = 23) mitotypes (herein) and the results of previous studies reporting similarly low frequencies within Pakistan agroecosystems  .The 3′‐mtB. tabaci mtCOI sequences archived in the JK Brown lab database (unpublished and published) were selected to represent the seven previously recognized major phylogeographic clades of B. tabaci  was carried out by the neighbor joining (NJ) method, implemented in Mega v6  to create a single haplotype sequence, and of these, one haplotype per whitefly was subjected to NJ analysis.Potential nuclear insertions of mitochondria (NUMTS) were detected by scanning mtThe optimal model of evolution was determined using jmodeltest software v2.1.7 . The 50% majority rule consensus tree was constructed with TreeAnnotator v1.8.4 . The mtCOI sequences were assigned to one of the seven major phylogeographic clades and a mitotype within  was carried out with BEAST v 1.8.4  sequence was PCR‐amplified using “universal” primers 27F (5′‐AGAGTTTGATCMTGGCTCAG) and 1513R  and Ars23S‐R . The PCR was set as described above using 20 ng of whitefly DNA. Cycling conditions were an initial denaturation at 95°C for 5 min, and 30 cycles of 95°C for 30 s, 60.5°C for 30 s, and 72°C for 45 s, with a final extension at 72°C for 10 min  were assembled using SeqMan Pro software available in DNASTAR Lasergene v8.0 . Reads and chromatograms were inspected for misalignment and manually edited to identify high‐quality contigs with no conflicts (0%) between forward and reverse reads. Sequences were exported in FASTA file format and aligned using the SILVA Incremental Aligner (SINA) v1.2.11, a ribosomal structure‐sensitive tool accessing more than two million bacterial reference sequences , classify.seqs, and removed.lineage algorithms, implemented in Mothur v1.43.0   . Selected reference sequences were B. tabaci‐Arsenophonus exemplars of the B. tabaci major clades, Asia I, Asia II, and Sub‐Saharan Africa. Sequences for B. tabaci and non‐B. tabaci were obtained from the JK Brown lab database or the GenBank database. The Arsenophonus outgroups selected were endosymbionts of the whiteflies, Aleurodicus dispersus (Russell), A. dugessi (Cockerell), Tetraleurodes acaciae (Quaintance), and Trialeurodes vaporariorum (West). The best‐fitting model was Tamura–Nei with gamma distribution rate variation and TrN + G+I (invariable sites). Bayesian analysis was carried out as described above.The 33.1COI sequences for whitefly B. tabaci field collections indicated they represented mitotypes that grouped in the major clade Asia II  relative frequency. The second most prevalent was Asia II‐7 occurring in collections from Lahore (urban area), at 8.3% (n = 23) frequency. The Asia II‐5 mitotype occurred at the lowest frequency among the three mitotypes identified, at 1.1% (n = 3), making it the least represented mitotype in Punjab province , the obligate primary endosymbiont, Arsenophonus (Alphaproteobacteria), one of two known mitotype‐specific major secondary endosymbionts, and Cardinium (Bacteroidia), a minor secondary endosymbiont known to occur in many other insects (arthropods), including B. tabaci.Among the most prevalent genera Tables  and S2 wPortiera and Arsenophonus occurred at a frequency of 100% and  was the second most abundant secondary endosymbiont. Though it was commonly associated with Asia II‐1 and Asia II‐5 mitotypes, it was conspicuously absent (undetectable) from the Asia II‐7  from Lahore, Lodhran, Okara, Rahim Yar Khan, and Vehari, Hemipteriphilus occurred at a frequency ranging from 5% to 20%, while infection of Asia II‐7 (n = 23) occurred at a frequency of 39%, while it was not detected at all the Asia II‐5 mitotype. Finally, Rickettsia was identified in the Asia II‐1 mitotype only and at a frequency of 2% in the individuals from Lahore, Okara, and Rahim Yar Khan  and Portiera–Arsenophonus–Cardinium (P‐A‐C) assemblages were most prevalent, at a 57% and 27% frequency, respectively. The assemblage consisting of P‐A predominated in whiteflies from the Sahiwal and Okara districts and were present in all three mitotypes at a frequency of 100% and 40%, respectively, and to lesser extents in all mitotypes from the other cotton‐growing districts of Punjab province  harbored P‐A and P‐A‐Hemipteriphilus (P‐A‐H) assemblages at a frequency of 61 and 39%, respectively. Despite an extensive number of samples tested (n = 250), the P‐A‐H assemblage was detected in only 2% of Asia II‐1 samples. The P‐A‐H together with either Cardinium (P‐A‐H‐C), Rickettsia (P‐A‐H‐R), Wolbachia (P‐A‐H‐W), or Cardinium with Rickettsia (P‐A‐H‐C‐R) were detected at a frequency of 0.4, 0.8, 1.2, and 0.8%, respectively. The P‐C‐Wolbachia (P‐C‐W) assemblage occurred in Asia II‐5 at a frequency of 33%, while the Asia II‐1 mitotype from Okara district harbored P‐A‐Rickettsia (P‐A‐R), at a frequency of only 3%  for the dendrogram favoring a close relationship of Asia II‐1 and II‐7, with Asia II‐5 being an outlier . The tree structure for clades I through V  and II‐7 (n = 2). In contrast, Arsenophonus OTUs 024 and 040 were rare, grouped in clades III and V, respectively, and occurred only in Asia II‐5 mitotypes (n = 2) from Lahore. In both instances, they diverged from the most abundant Arsenophonus species by 3%–7%  and A. dugessi (Accession no. AF286129) [subfamily Aleurodicinae] grouped in the basal position of the tree and diverged by 3%–8% from Arsenophonus OTUs associated with the Aleyrodinae (subfamily), perhaps indicative of subfamily specific OTU barriers  within of the Punjab district, suggests that certain mitotype–endosymbiont communities may have been/be selectively associated with previous and current outbreaks of cotton leaf curl disease, respectively. The leaf curl disease is caused by approximately ten endemic, divergent begomoviral species, and strains (begomovirome) that cycle in cultivated and wild plant hosts in the agroecosystem, as single infections or as mixtures from which viral recombinants emerge and spread throughout the region  of this symbiont is not exclusive to 4.2B. tabaci mitotype dynamics have provided insights into population dynamics, geographic distribution, and genetic/genomic structure of the B. tabaci group, a group as a whole for which most biological, genetic, and population‐level features are poorly understood  and evolutionary (phylogenetic) considerations are relatively informative. Based on endosymbiont composition, the phylogenetically confounded Asia II‐1 and Asia II‐7 “species”  and the number and type of shared OTUs , with 12 of the 13 bacterial groups having been documented. This was followed by the Lahore whitefly samples that harbored six of the 13 groups. The climate microniches in Okara and Lahore are humid and subtropical, while by comparison, the other districts are characterized as either an arid desert or semi‐arid  and thereby alter the dynamics of virome composition due to mitotype‐specific transmission competency.Of interest, among the distribution patterns reported herein are that spread of Arsenophonus OTUs  and phylogenetic analyses (Bayesian and maximum likelihood) indicated all three Asia II mitotypes harbored six distinct Arsenophonus OTUs have been reported from Asia II samples in India  for certain mitotypes may have resulted in biased estimated frequencies of detectable (or undetectable) OTUs. In particular, Cardinium, identified from a number of mitotypes worldwide, may occur at low, undetectable levels, and if so, studying a larger population size may show it also occurs in Asia II‐1 or II‐5 mitotypes. Infection of the polyphagous A mitotype of B. tabaci by Cardinium has previously been associated with increased fecundity  than Asia II‐1 (n = 250) mitotypes, at a 39% and 6% frequency, respectively. The relatively high abundance of Hemipteriphilus in Lahore, where urban landscapes are abundant and pesticide use is rare, suggests this endosymbiont may have an important role in mitotype adaptability to this and perhaps other micro‐environmental niches.Finally, the frequency of B. tabaci mitotypes that could greatly improve whitefly management strategies by associating OTU assemblages, mitotypes, and CLCuD‐begomoviral species and strains with outbreaks. Routine surveillance and mapping would provide an early warning system such that precautions associated with the different management practices suspected of exacerbating CLCuD outbreaks, including shifting cotton genotypes with no knowledge of resistance to specific virus strain or species, and insecticide programs that stimulate whitefly upsurgences among particular mitotypes or others.Together, these results highlight the importance of routine monitoring of endosymbiont OTUs and the associated 5Portiera and secondary endosymbiont Arsenophonus were fixed in the Asia II populations. In contrast, Cardinium was detected in 29% of B. tabaci sampled, whereas Hemipteriphilus, Rickettsia, and Wolbachia were detected rarely, making them minor endosymbionts, at a 2%–8% frequency. Collectively, at least 43 OTUs taxonomically classified bacterial genera comprised the microbiome of the three Asia II whitefly mitotypes.The primary endosymbiont Arsenophonus, Cardinium, Hemipteriphilus, and Wolbachia between the Asia II mitotypes where their ranges overlapped in Lahore and that certain Arsenophonus OTUs were uniquely present in one mitotype over the other. Also, based on the high fixation rate of Arsenophonus, it is tempting to speculate the occurrence of a selective sweep by the widely prevalent OTU assemblage in parallel with/leading up to the recent “recovery” observed for Asia II‐1 in the cotton agroecosystems , immediately before and during the time this study was conducted.The results predict horizontal transfer has occurred for several unique OTUs of Cardinium and/or Rickettsia in the geographic and genetic demographic expansions resulting from an increase in female offspring production concurrent with enhanced insecticide resistance, respectively (Caballero, Although somewhat speculative, the whitefly expansion may also have contributed to the virus outbreak if the Asia II‐1 is found to be a preferentially competent vector of CLCuKoV‐Bur, over other known vector mitotype–begomoviral combinations of CLCuD species or strains in the Punjab (Pan, Cui, & Chen, B. tabaci mitotypes studied here and also for future studies of large‐scale relationships among global mitotypes.Finally, also of interest is that the composition of OTU assemblages that associate with different mitotypes can provide a useful tool and indicator of genetic/genomic relatedness among the Asia II The authors declare no conflict of interest.J.R.P.‐M. performed analyses, wrote the first draft, and contributed to methodology, review and editing. M.Z.‐U.‐R., U.H., and M.S.H. conducted fieldwork, collected data, contributed to methodology, and data curation. H.‐W.H. contributed to conceptualization, methodology and review. J.K.B. conceived the ideas, and contributed to methodology, resources, funding acquisition, supervision, review and editing. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.