Mycoplasma species in vaginas of dairy cows before and after exposure to bulls and their association with conception.

Mycoplasma species can colonize the urogenital tract of dairy cattle. However, interrelationships between Mycoplasma spp. and reproductive performance in dairy herds are unclear. In this study, we measured apparent prevalences of Mycoplasma spp. in the vaginas of dairy cows (n = 629) pre- and post-bull exposure in dairy herds with and without Mycoplasma bovis clinical disease (n = 5 herds), and assessed associations between variables describing reproductive performance and consequent Mycoplasma spp. isolation. Mycoplasma spp. were infrequently isolated from the vagina pre- (1.9%; 12/629) and post-bull (3.2%; 20/629) exposure. Of the mycoplasmas isolated, Mycoplasma bovigenitalium was isolated most frequently (87.5%; 28/32), followed by Mycoplasma californicum (9.3%; 3/32). Mycoplasma bovis was only isolated from one cow. We were unable to provide any evidence of venereal transmission of M. bovis in cows in M. bovis-infected herds that use natural service bulls. There was an insufficient number of cows with Mycoplasma spp. in the vagina pre-bull exposure to assess effects on subsequent reproductive performance. Cows that had not conceived before post-bull exposure sampling had much greater odds (odds ratio 14.8; 95% confidence interval 4.2 to 52.3) of having a Mycoplasma sp. isolated from the vagina at this time compared with those that had conceived. Also, within those that had conceived, delayed conception increased the odds of having a Mycoplasma spp. isolated from the vagina at the post-bull exposure sampling by a factor of 1.62 for every additional week not pregnant. The likely cause of these findings is that cows that remain not pregnant for longer are more likely to be served by a bull (likely repeatedly) and subsequently become colonized with a Mycoplasma sp. (mostly M. bovigenitalium) through venereal transmission. In dairy herds that use bulls, there is a greater chance of isolating a Mycoplasma sp. (mostly M. bovigenitalium) after a period of bull breedings from the vaginas of cows that have remained nonpregnant for longer during the bull breeding period.

Mycoplasma bovis and other Mollicutes in replacement dairy heifers from Mycoplasma bovis-infected and uninfected herds: A 2-year longitudinal study.

Replacement dairy heifers exposed to Mycoplasma bovis as calves may be at risk of future clinical disease and pathogen transmission, both within and between herds; however, little information is available about these risks. We conducted a 2-yr longitudinal (panel) study starting with 450 heifer calves reared to weaning in 8 herds (7 M. bovis infected with clinical disease, 1 uninfected) under the same ownership. After weaning, heifers were commingled and managed with non-study heifers at a single heifer rearing facility. Nose, conjunctival, and vaginal swabs were collected along with a blood sample at weaning, prebreeding, precalving, and approximately 1 mo postcalving. Additionally, a colostrum sample was collected upon calving and a composite milk sample was collected 1 mo postcalving. The swabs, colostrum, and milk samples were cultured for Mycoplasma spp., and serum from the blood was evaluated for serological evidence of exposure to M. bovis using an ELISA. Despite a high M. bovis ELISA seroprevalence at weaning in the heifers from the 7 M. bovis-infected herds with clinical disease [72% (289/400); range by herd: 28-98%], M. bovis was isolated from only 4% (16/400) of the same heifers at the same time. In heifers from the uninfected herd at weaning, M. bovis seroprevalence was 2% (1/50) and M. bovis was not detected by culture. Mycoplasma bovis was isolated from 0.5% (2/414) of heifers at prebreeding, 0% (0/374) of heifers at precalving, and 0.3% (1/356) of heifers 1 mo postcalving. The nose was the predominant anatomical site of M. bovis colonization (74%; 14/19 culture positives). A single heifer (from an M. bovis-infected herd with clinical disease) was repeatedly detected with M. bovis in its nose at weaning, prebreeding, and postcalving samplings. This demonstrates the possibility, albeit rare, of a long-term M. bovis carrier state in replacement heifers exposed to M. bovis as calves, up to at least 1 mo after entry into the milking herd. No M. bovis clinical disease was detected in any heifer from weaning through to the end of the study (approximately 1 mo after calving). Acholeplasma spp. were commonly isolated throughout the study. Mycoplasma bovigenitalium, Mycoplasma bovoculi, and Mycoplasma bovirhinis were isolated infrequently. Mycoplasma bovis seroprevalences at prebreeding, precalving, and postcalving samplings were 27% (112/414), 12% (46/374), and 18% (65/356), respectively. Overall, the results show that replacement heifers from groups exposed to M. bovis preweaning can become colonized with M. bovis and that colonization can, uncommonly, be present after their first calving. For groups of 50 or more heifers exposed to M. bovis preweaning, there is at least a nontrivial probability that the group will contain at least 1 shedding heifer postcalving.

Whole dairy herd sampling to detect subclinical intramammary Mycoplasma bovis infection after clinical mastitis outbreaks.

After clinical Mycoplasma bovis mastitis outbreaks in dairy herds, M. bovis can persist as subclinical intramammary infections. Identification and culling of sub-clinically infected cows may be warranted to reduce future pathogen transmission and disease. In this study, apparent cow-level prevalences of M. bovis intramammary infection within 4 milking herds immediately following outbreaks of clinical disease due to M. bovis were determined utilising PCR and culture. All clinically affected M. bovis cows had been culled from the herds prior to herd sampling. Composite milk samples were collected once from each cow (n = 2,258) using a routine milk recording sampling technique. These samples were pooled for PCR screening; positive pools were analysed in different sized pools as needed from large to small, until individual PCR-positive animals could be identified. Despite M. bovis seroprevalences of 76% (herd 1), 40% (herd 2), 20% (herd 3) and 16% (herd 4), apparent prevalences of intramammary infection in the main milking group based on PCR in herds 1 to 4 were 0.2% (1/497), 0.0% (0/475), 0.1% (1/816) and 0.0% (0/444), respectively. Due to the low apparent prevalences of subclinical intramammary mycoplasma infections in these herds and the high expense associated with milk sample collection and testing, the return on diagnostic investment was very limited, particularly considering that additional cows are likely to have been colonised with mycoplasma in other anatomical sites. The results of this study suggest that pursuing identification of cows with subclinical intramammary mycoplasma infections following resolution of clinical M. bovis disease outbreaks in dairy herds may be of minimal benefit in programs designed to control or eradicate M. bovis.

Isolation of Mycoplasma spp. and serological responses in bulls prior to and following their introduction into Mycoplasma bovis-infected dairy herds.

With the common use of bulls for breeding following a period of artificial insemination in seasonally bred dairy herds, it is important to consider the potential role of the bull in transmission of Mycoplasma spp. within and between herds. This study aimed to assess the prevalence of Mycoplasma spp. in a population of bulls before and after use in Mycoplasma bovis-infected herds. The frequency of subclinical infection was also measured serologically postbreeding, and the association of Mycoplasma spp. on semen quality was evaluated. Mycoplasma bovis was isolated from 4 of 118 bulls after use in 4 herds infected with M. bovis. In the bulls, M. bovis seroprevalence increased from 9% prebreeding to 46% postbreeding with a total seroconversion rate of 44% across the 4 herds, with no evidence of clinical disease. There was no association of Mycoplasma spp. in the bulls' semen and abnormal palpation characteristics (enlarged or nodular) of seminal vesicular glands or poor semen quality attributes such as semen mass activity, sperm motility, and morphology. These results demonstrate a high degree of subclinical exposure of the bulls to M. bovis in infected herds and highlight the potential for bulls to be mycoplasma carriers within and between herds. Herd biosecurity protocols and control programs should take into account the potential role of bulls in the introduction and spread of Mycoplasma spp.

Short communication: Shedding of Mycoplasma bovis and antibody responses in cows recently diagnosed with clinical infection.

Mycoplasma bovis can have significant consequences when introduced into immunologically naïve dairy herds. Subclinically infected carrier animals are the most common way that M. bovis is introduced into herds. Although M. bovis udder infections can be detected by milk sampling lactating animals before their introduction, currently, no definitive way of identifying M. bovis carrier animals that are nonlactating (i.e., calves, heifers, dry cows, or bulls) is available. Understanding the prevalence of M. bovis shedding from various body sites in clinically infected animals could inform strategies for the detection of subclinical infection in nonlactating stock. The mucosal surfaces of the nose, eye, and vagina of 16 cows with recent clinical mastitis caused by M. bovis were examined for the presence of M. bovis shedding. Blood was collected for serological evaluation by a commercially available ELISA. Mycoplasma bovis was isolated from the vagina of only 3 (18.8%) of the cows and was not detected from the noses or eyes of any of the cows. Fifteen of the 16 (93.8%) cows were seropositive to the ELISA. With such low prevalence of detection of M. bovis from the vagina and no detections from the noses or eyes of recently clinically infected animals, it is very likely that sampling these sites would be ineffective for detecting subclinical infection in cattle. Serology using the ELISA may have some use when screening animals for biosecurity risk assessment. However, more information regarding time to seroconversion, antibody longevity, and test diagnostic sensitivity and specificity are required to define the appropriate use of this ELISA for biosecurity purposes.

Bulk tank milk antibody ELISA as a biosecurity tool for detecting dairy herds with past exposure to Mycoplasma bovis.

In Australia, one of the biosecurity recommendations to help prevent the introduction of Mycoplasma bovis into a dairy herd is to use a PCR assay on bulk tank milk (BTM) samples to evaluate the M. bovis infection status of potential source herds. An alternative approach is to assess the immunological status of the herd with respect to previous exposure to M. bovis via the use of an ELISA that is commercially available for use on cattle milk and serum. The objectives of this study were to (1) evaluate factors potentially associated with variation in the ELISA BTM optical density coefficient (ODC%) in previously exposed herds, (2) evaluate the association between the proportion of cows that are ELISA positive and the BTM ELISA ODC%, (3) assess agreement between the BTM ELISA and PCR and culture, and (4) compare BTM ELISA ODC% between the "hospital" herd and the main lactating herd on the same farm. Bulk tank milk samples (n = 192) were collected from 19 dairy herds with a history of clinical M. bovis disease and from 6 control herds (herds with no known clinical cases of mycoplasmosis). For 28 of the BTM samples collected, blood was also collected from 50 lactating cows contributing to that bulk tank sample. From 1 herd, concurrent paired BTM samples were collected from the main herd and the hospital herd on 16 occasions. All BTM samples were analyzed by ELISA (Bio-X Bio K 302, Bio-X Diagnostics, Rochefort, Belgium), PCR, and culture. The BTM ELISA ODC% was associated with time since initial M. bovis outbreak and time since the start of the herd's calving period. Following an initial outbreak of M. bovis, the BTM ELISA ODC% was highest in the first 8 mo. In split- and seasonal-calving herds, significantly higher BTM ELISA ODC% results were observed 5 to 8 wk after the commencement of the calving period. A significant association was observed between the within-herd seroprevalence for the lactating herd and BTM ELISA ODC%, but within-herd seroprevalence explained little of the variation in BTM ELISA ODC%. When comparing the BTM ELISA with a multiplex probe PCR and culture followed by 16S to 23S rRNA sequencing, there was virtually no agreement above that expected by chance; prevalence-adjusted bias-adjusted kappa values were 0.22 and 0.25 for ELISA category versus PCR category and culture, respectively. Finally, the hospital herd BTM ELISA ODC% mirrored that for the main herd BTM but was significantly higher. This study demonstrates that this commercially available ELISA used on BTM samples may complement the use of BTM PCR or culture in identifying herds from which purchase of animals may pose a higher biosecurity risk for introduction of M. bovis into noninfected herds.

Milk acidification to control the growth of Mycoplasma bovis and Salmonella Dublin in contaminated milk.

Bacterial contamination of milk fed to calves compromises calf health. Several bacterial pathogens that infect cows, including Mycoplasma bovis and Salmonella enterica ssp. enterica serovar Dublin, are shed in milk, providing a possible route of transmission to calves. Milk acidification lowers the milk pH so that it is unsuitable for bacterial growth and survival. The objectives of this study were to (1) determine the growth of M. bovis and Salmonella Dublin in milk, and (2) evaluate the efficacy of milk acidification using a commercially available acidification agent (Salstop, Impextraco, Heist-op-den-Berg, Belgium) to control M. bovis and Salmonella Dublin survival in milk. For the first objective, 3 treatments and a positive control were prepared in 10 mL of milk and broth, respectively, and inoculated with M. bovis or Salmonella Dublin to an approximate concentration of 104 cfu/mL. Each treatment was retained at 5, 23, or 37°C with the positive control at 37°C. Aliquots were taken at 4, 8, 24, 28, 32, 48, 52, and 56 h after inoculation and transferred onto agar medium in triplicate following a 10-fold dilution series in sterile phosphate-buffered saline. All plates were incubated and colonies counted. For the second objective, 4 treatments and a positive control were prepared with 100 mL of milk and inoculated with M. bovis or Salmonella Dublin to an approximate concentration of 106 cfu/mL. With the use of Salstop, treatments were adjusted to an approximate pH of 6, 5, 4, or 3.5. The positive control was left untreated. At 1, 2, 4, 6, 8, and 24 h after treatment, triplicate aliquots were taken, the pH measured, and then the aliquots were transferred onto agar medium and into broth for enrichment. Following incubation, agar colonies were counted, while broths were plated and incubated prior to colonies being counted. All trials were repeated. Mycoplasma bovis did not grow in milk, but Salmonella Dublin proliferated. The pH of all acidification treatments remained stable for 24 h. No viable M. bovis organisms were detected at 1 h of exposure to pH 3.5 and 4 or at 8 h of exposure to pH 5. Following 24 h of exposure to pH 6 M. bovis remained viable. No viable Salmonella Dublin organisms were detected at 2 and 6 h of exposure to pH 3.5 and 4, respectively. Salmonella Dublin remained viable following 24 h of exposure to pH 5 and 6. These results demonstrate that milk acidification using Salstop is effective at eliminating viable M. bovis and Salmonella Dublin organisms in milk if the appropriate pH and exposure time are maintained.

Low serum and serum-free culture of multipotential human adipose stem cells.

BACKGROUND: Adipose tissue provides an easily accessible and abundant source of putative stem cells for translational clinical research. Currently prevalent culture techniques include the use of FBS, a highly variable and undefined component, which brings with it the potential for adverse patient reactions. In an effort to eliminate the use of animal products in human adipose stem cell (ASC) cultures, we have developed two new culture methods, a very low human serum expansion medium and a completely serum-free medium. METHODS: Through serial testing, a highly enriched medium formulation was developed for use with and without the addition of 0.5% human serum, an amount easily obtainable from autologous blood draws. RESULTS: Very low-serum culture yielded population-doubling times averaging 1.86 days in early passage, while the serum-free formulation was associated with less robust cell growth, with doubling times averaging 5.79 days. ASC in both conditions maintained its ability to differentiate into adipo-, chondro- and osteogenic lineages in vitro, despite lower expression of CD34 in early passage. Expression of ALDH, HLA, CD133, CD184, and CD31 was comparable with that seen in cells cultured in 10% FBS. DISCUSSION: These newly developed culture conditions provide a unique environment within which to study ASCs without the interference of animal serum, and allow for the rapid expansion of autologous ASCs in culture in an animal product-free environment for use in human clinical trials.

A six-year prospective study of pathways toward drug use in adolescent boys with and without a family history of a substance use disorder.

OBJECTIVE: The purpose of this study was to test a developmental model of drug use in male adolescents. The model postulates that low executive functioning and a difficult temperament are related to aggression and affiliations with delinquent peers which, in turn, are related to elevated drug use. METHOD: Boys (N = 187) with and without a family history of a substance use disorder (SUD) were followed over a 6-year period. Executive functioning and temperament were measured at age 10-12, aggression and affiliations with delinquent peers were assessed at age 12-14 and drug use was measured at age 16. RESULTS: Low executive functioning and a difficult temperament were related to increased aggression and affiliations with delinquent peers. These latter variables were related to increased drug use. Furthermore, the relation between difficult temperament and drug use was fully mediated by aggression and affiliations with delinquent peers. CONCLUSIONS: Drug abuse prevention efforts may benefit from clinical interventions aimed at strengthening executive functioning, regulating temperament and improving socialization strategies in antisocial children.

Psychopathology and substance-related problems during early adolescence: a survival analysis.

Examined the chronological and statistical relations among onsets of psychopathology, alcohol and cannabis use, and substance-related problem psychopathology, alcohol and cannabis use, and substance-related problems from late childhood through early adolescence in boys of fathers with substance use disorder (SUD; high average risk: n = 177) and without SUD (low average risk: n = 203) using survival analysis. Proportional hazard models indicated that antisocial disorders were predicted by risk group and mediated the observed relation between risk group and substance-related problems. Negative affect disorders were predicted by risk group but did not predict substance involvement in early adolescence. Results support a model in which paternal SUD predisposes to increased antisocial and negative affect disorders in boys, and antisocial disorders lead to substance-related problems in early adolescence.

Possible association of influenza A with fetal loss: investigation of a cluster of spontaneous abortions and stillbirths.

It has been suspected that influenza infection is associated with fetal or perinatal mortality, but little recent evidence supports this hypothesis. A small cluster of early and late fetal deaths in early 1986 prompted an epidemiological investigation. Women whose pregnancies were affected (cases) were compared with women whose pregnancies had a normal outcome (controls). Case pregnancies were distinguished by a significant excess of recent flu-like illness (p = 0.006), and were significantly more likely than controls to have serological evidence of influenza A infection (p = 0.00067), predominantly the influenza A H3N2, Christchurch/4/85-like strain. The cluster was recognised because most cases were patients of one health centre. Larger epidemiological studies will be needed to confirm an association between influenza A and fetal death, but this cluster suggests that influenza A may have an adverse influence on fetal survival.