Mycoplasma agalactiae ST35: a new sequence type with a minimal accessory genome primarily affecting goats.

BACKGROUND: Mycoplasma agalactiae, causing agent of contagious agalactia, infects domestic small ruminants such as sheep and goats but also wild Caprinae. M. agalactiae is highly contagious and transmitted through oral, respiratory, and mammary routes spreading rapidly in an infected herd. RESULTS: In an outbreak of contagious agalactia in a mixed herd of sheep and goats, 80% of the goats were affected displaying swollen udders and loss of milk production but no other symptom such as kerato-conjunctivitis, arthritis or pulmonary distress commonly associated to contagious agalactia. Surprisingly, none of the sheep grazing on a common pasture and belonging to the same farm as the goats were affected. Whole genome sequencing and analysis of M. agalactiae strain GrTh01 isolated from the outbreak, revealed a previously unknown sequence type, ST35, and a particularly small, genome size of 841'635 bp when compared to others available in public databases. Overall, GrTh01 displayed a reduced accessory genome, with repertoires of gene families encoding variable surface proteins involved in host-adhesion and variable antigenicity being scaled down. GrTh01 was also deprived of Integrative Conjugative Element or prophage, and had a single IS element, suggesting that GrTh01 has a limited capacity to adapt and evolve. CONCLUSIONS: The lack of most of the variable antigens and the Integrative Conjugative Element, both major virulence- and host specificity factors of a M. agalactiae strain isolated from an outbreak affecting particularly goats, indicates the implication of these factors in host specificity. Whole genome sequencing and full assembly of bacterial pathogens provides a most valuable tool for epidemiological and virulence studies of M. agalactiae without experimental infections.

The prevalence of Coxiella burnetii shedding in dairy goats at the time of parturition in an endemically infected enterprise and associated milk yield losses.

BACKGROUND: This was a panel study of the prevalence of C. burnetii infection in does in an endemic dairy goat enterprise in Victoria, Australia. Our first objective was to determine the prevalence of does shedding C. burnetii at the time of parturition and to quantify the concentration of genome equivalents (GE) present in each C. burnetii positive sample. Our second objective was to determine the proportion of positive does that were persistent shedders. Our final objective was to quantify the association between C. burnetii qPCR status at the time of kidding and daily milk volumes produced during the subsequent lactation. RESULTS: Vaginal swabs (n= 490) were collected from does at the time of kidding and analysed using a quantitative polymerase chain reaction (qPCR) assay. Shedding of C. burnetii was detected in 15% (95% CI: 12% to 18%) of the sampled does. Does were classified as qPCR-negative, qPCR-positive low and qPCR-positive high based on the estimated concentration of GE from the qPCR. Persistent shedding at relatively low concentrations was detected in 20% (95% CI: 10% to35%) of shedding does sampled again at their subsequent parturition. After controlling for possible confounders and adjusting for variation in daily milk yields at the individual doe level, daily milk yields for qPCR-positive high does were reduced by 17% (95% CI: 3% to 32%) compared to qPCR-negative does (p= 0.02). CONCLUSIONS: Shedding concentrations of C. burnetii were highly skewed, with a relatively small group of does shedding relatively high quantities of C. burnetii. Further, high shedding does had reduced milk yields compared to qPCR-negative does. Early detection and culling of high shedding does would result in increased farm profitability and reduce the risk of Q fever transmission.

Synthesis of Recombinant P48 of Mycoplasma agalactiae by Site Directed Mutagenesis and its Immunological Characterization.

Contagious agalactia caused by Mycoplasma agalactiae is an economically important disease of sheep and goats and has been prevalent worldwide including India. The present study was undertaken to evaluate the membrane protein P48 of M. agalactiae for specific diagnosis of disease. For this, p48 gene of the organism was amplified by PCR and subjected to site directed mutagenesis to convert three TGA codons to TGG's and, subsequently, cloned into prokaryotic expression vector pPRO EX HTb. Purified recombinant P48 protein reacted to anti-P48 serum in western blotting, which confirmed its immunogenic nature. Furthermore, the immune-blotting of the cell lysates from various Indian isolates of M. agalactiae against anti-P48 serum resulted in a single band at âˆ¼ 48 kDa among all isolates, indicating the conserved nature of P48 antigen in M. agalactiae. Also, the cross reactivity of P48 antigen among various Mycoplasma spp. was checked by western blotting which revealed reactivity only with M. agalactiae and M. bovis. Hence, this antigen could be exploited to differentiate M. agalactiae from other pathogenic Mycoplasma species except M. bovis. However, the inability of P48 to distinguish M. agalactiae from M. bovis does not downgrade the significance of P48 as the two species are usually host specific.

Contagious agalactia due to Mycoplasma spp. in small dairy ruminants: epidemiology and prospects for diagnosis and control.

Contagious agalactia (CA) is a serious disease of small dairy ruminants that has a substantial economic impact on the goat and sheep milk industries. The main aetiological agent of the disease is Mycoplasma agalactiae, although other species, such as Mycoplasma mycoides subsp. capri, Mycoplasma capricolum subsp. capricolum and Mycoplasma putrefaciens, are pathogenic in goats. There are two clinical-epidemiological states of CA in sheep and goats; herds and flocks may exhibit outbreaks of CA or may be chronically infected, the latter with a high incidence of subclinical mastitis and only occasional clinical cases. The complex epidemiology of CA is related to the genetic characteristics and mechanisms of molecular variation of the Mycoplasma spp. involved, along with presence of CA-mycoplasmas in wild ruminant species. In goats, the situation is particularly complex and asymptomatic carriers have been detected in chronically infected herds. The coexistence of other non-pathogenic mycoplasmas in the herd further complicates the diagnosis of CA and the design of efficient strategies to control the disease. Routes of infection, such as the venereal route, may be involved in the establishment of chronic infection in herds. Current challenges include the need for improved diagnostic methods for detection of chronic and subclinical infections and for the design of more efficient vaccines.

The role of bacterial pathogens in coliform mastitis in sows.

Even in modern piglet production, mastitis and lactation failure in sows represent a considerable health problem post partum, affecting in its consequences both the sow and her piglets. Known as a multifactorial syndrome, Mastitis-Metritis-Agalactia (MMA) has been topic of several studies investigating possible influencing factors at farm and sow level in the recent past. However, there is a lack of current investigations on the causative pathogens, especially with advanced laboratory methods and with an adequate control group of healthy animals. Therefore, 1026 milk samples from coliform mastitis (CM)-affected, and 972 samples from healthy sows on six farms were examined bacteriologically in this study. The spectrum of isolated bacteria did not differ significantly between diseased and healthy animals for most species, with Escherichia coli as predominant species with 70.4% positive samples from diseased, and 78.0% positive samples from healthy animals. Furthermore, other Enterobacteriaceae, Staphylococcaceae, Streptococcaceae and Enterococcaceae were isolated both from CM-affected and non-affected animals.The similar bacteria distribution underlines the multifactorial pathogenesis of CM: Only with further adverse--endogen or exogen--factors being present, ubiquitous bacteria from the sow's environment can contribute to the development of clinical signs of infection.

Effects of intramammary infections on somatic cell score and milk yield in Sarda sheep.

AIM: To evaluate the effects of intramammary infections (IMI) on somatic cell score (SCS) and milk yield in dairy ewes. METHODS: Monthly milk samples were collected from a flock of 202 Sarda sheep, over a period of 7 months, for bacteriological culture and measurement of somatic cell counts (SCC). During the same period, milk yield was measured daily using electronic milk meters connected to each half-udder cluster of the milking machine. SCC was transformed to SCS using a base-2 log transformation. One SCS is equivalent to a SCC of 25,000 cells/ml, and each increase of 1 in SCS is associated with doubling of the SCC. IMI was defined by the presence of five or more colonies of similar morphology isolated from a milk sample (≥500 cfu/ml). The effect of IMI on SCS and milk yield was assessed using a generalised estimating equation (GEE). RESULTS: There were 1,186 udder halves with IMI from 2,828 milk samples, a prevalence of 41.9%. The distribution of bacterial species within the 1,186 culture-positive samples was comprised of 476 (40.1%) Staphylococcus epidermidis, 172 (14.5%) Staph. chromogenes, 38 (3.2%) Staph. caprae, 134 (11.3%) Staph. simulans, 114 (9.6%) Streptococcus uberis, 123 (10.4%) Strep. dysgalactiae, and 129 (10.9%) Strep. equi subsp. zooepidemicus. SCS was greater in udder halves with IMI (mean 7.71; SD 0.82) than in udder halves without IMI (mean 5.53; SD 1.02) (p<0.01). IMI due to streptococcal species were associated with greater SCS (mean 8.24; SD 0.62) than those due to staphylococcal species (mean 7.48; SD 0.79) (p<0.01). Milk yield from udder halves with IMI was lower (mean 439 (SD 162) ml/half udder/day) than from udder halves without IMI (mean 602 (SD 170) ml/half udder/day) (p<0.01). IMI due to staphylococcal species was associated with a lower milk yield (mean 399 (SD 167) ml/half udder/day) than IMI due to streptococcal species (mean 427 (SD 156) ml/half udder/day) (p<0.01). CONCLUSIONS: These findings provide sheep milk producers with information on the losses associated with subclinical mastitis, which can be used to evaluate the economics of prevention and treatment protocols concerning udder health in ovine dairy flocks.

Bacteria in milk from anterior and posterior mammary glands in sows affected and unaffected by postpartum dysgalactia syndrome (PPDS).

BACKGROUND: The performance of piglet weight gain is strongly dependent on the sow's ability to meet the demand for adequate milk. Postparturient disorders, especially those subsumed under the term postpartum dysgalactia syndrome (PPDS), can alter or reduce the milk production sensitively, resulting in starving piglets. The aim of this study was to gather further information about the prevalence of different bacterial species in the anterior and posterior mammary glands of sows with respect to the clinical appearance of PPDS. METHODS: In this study, the health status of 56 sows after farrowing was determined with special regard to mastitis and dysgalactia. Pooled milk samples from anterior and posterior glands were taken from both affected and non-affected animals and analysed bacteriologically for the presence of a wide spectrum of different pathogens. RESULTS: Mainly Escherichia coli, staphylococci and streptococci were detected in high percentages but without significant differences in healthy and diseased animals and anterior and posterior glands. However, the large percentages of coliform bacteria suggested a transmission route via faecal contamination. CONCLUSION: In this study, the prevalence of different bacteria in anterior and posterior glands in PPDS positive and negative sows was analysed. No significant differences in bacteria of healthy and diseased sows were assessed. Therefore, the development of clinical PPDS and actual infection seems to be largely dependant on individual resistance in single sows.

Effect of feeding sorghum ergot (Claviceps africana) to sows during mid-lactation on plasma prolactin and litter performance.

Diets containing 3% sorghum ergot (16 mg alkaloids/kg, including 14 mg dihydroergosine/kg) were fed to 12 sows from 14 days post-farrowing until weaning 14 days later, and their performance was compared with that of 10 control sows. Ergot-fed sows displayed a smaller weight loss during lactation of 24 kg/head vs. 29 kg/head in control sows (p > 0.05) despite feed consumption being less (61 kg/head total feed intake vs. 73 kg/head by control sows; p < 0.05). Ergot-fed sows had poorer weight gain of litters over the 14-day period (16.6 kg/litter vs. 28.3 kg/litter for controls; p < 0.05) despite an increase in consumption of creep feed by the piglets from the ergot-fed sows (1.9 kg/litter compared with 1.1 kg/litter by the control; p > 0.05). Sow plasma prolactin was reduced with ergot feeding after 7 days to 4.8 microg/l compared with 15.1 microg/l in the control sows (p < 0.01) and then at weaning was 4.9 microg/l compared with 8.0 microg/l (p < 0.01) in the control sows. Two sows fed ergot ceased lactation early, and the above sow feed intakes, body weight losses with litter weight gains and creep consumption indirectly indicate an ergot effect on milk production.

Feeding sorghum ergot (Claviceps africana) to sows before farrowing inhibits milk production.

OBJECTIVE: To assess the impact of feeding different amounts of sorghum ergot to sows before farrowing. DESIGN: Fifty-one pregnant sows from a continually farrowing piggery were sequentially inducted into the experiment each week in groups of four to seven, as they approached within 14 days of farrowing. Diets containing sorghum ergot sclerotia within the range of 0 (control) up to 1.5% w/w (1.5% ergot provided 7 mg alkaloids/kg, including 6 mg dihydroergosine/kg) were randomly allocated and individually fed to sows. Ergot concentrations were varied with each subsequent group until an acceptable level of tolerance was achieved. Diets with ergot were replaced with control diets after farrowing. Post-farrowing milk production was assessed by direct palpation and observation of udders, and by piglet responses and growth. Blood samples were taken from sows on three days each week, for prolactin estimation. RESULTS: Three sows fed 1.5% ergot for 6 to 10 days preceding farrowing produced no milk, and 87% of their piglets died despite supplementary feeding of natural and artificial colostrums, milk replacer, and attempts to foster them onto normally lactating sows. Ergot inclusions of 0.6% to 1.2% caused lesser problems in milk release and neo-natal piglet mortality. Of 23 sows fed either 0.3% or 0.6% ergot, lactation of only two first-litter sows were affected. Ergot caused pronounced reductions in blood prolactin, and first-litter sows had lower plasma prolactin than multiparous sows, increasing their susceptibility to ergot. CONCLUSION: Sorghum ergot should not exceed 0.3% (1 mg alkaloid/kg) in diets of multiparous sows fed before farrowing, and should be limited to 0.1% for primiparous sows, or avoided completely.

In vitro susceptibilities of field isolates of Mycoplasma mycoides subsp. mycoides large colony type to 15 antimicrobials.

In vitro susceptibilities of 16 Mycoplasma mycoides subsp. mycoides large colony type field isolates to 15 antimicrobial agents were determined using a broth microdilution method. The most effective antimicrobials were fluoroquinolones, tetracyclines and macrolides, with MIC values under 2 microg/ml. Resistance to nalidixic acid, gentamicin, streptomycin and spectinomycin was observed.

Factors of variation influencing bulk tank somatic cell count in dairy sheep.

Between January and December 2002, a total of 21,685 records for bulk tank milk somatic cell count (BTSCC) were obtained from 309 dairy ewe herds belonging to the Sheep Improvement Consortium in Castilla-Leon, Spain. Based on the first statistical model, ANOVA detected significant effects of herd, breed, month within herd, dry therapy, type of milking, contagious agalactia, and installations within machine milking on logBTSCC. A second statistical model was used on herds with machine milking to study the effect of the vacuum level and pulsation rate on BTSCC. Herd and month within herd were important variation factors as they explained 48.4 and 16.1% of the variance in BTSCC. Variability in logBTSCC among breeds ranged from 5.84 (Castellana) to 6.09 (Awassi and Spanish Assaf). Implementing dry-ewe therapy (5.91) significantly reduced logBTSCC compared with when it was not implemented (6.10). Hand milking elicited greater logBTSCC (6.07) than machine milking (5.94). Machine milking of ewes in milking parlors (logBTSCC: 5.88 to 5.94) was associated with better udder health than was the use of bucket-milking machines (6.04). Reduced vacuum levels and elevated pulsation rate during machine milking optimized BTSCC. In all cases, clinical outbreaks of contagious agalactia increased BTSCC. As a result, dry therapy was proposed as the main tool to reduce BTSCC. Optimization of milking-machine standards and parlor systems also improved udder health in dairy sheep.

Serological study of contagious agalactia in herds of goats in the Canary Islands.

An indirect ELISA, using local strains of Mycoplasma agalactiae and Mycoplasma mycoides subspecies mycoides large colony (MmmLC), was applied to evaluate the seroprevalence of M agalactiae and MmmLC in flocks of goats on each of the Canary Islands. In total 3890 samples of serum were collected from 204 flocks. The results indicated that the seroprevalence of both organisms is high on all the islands; average values of 55 per cent and 67 per cent were recorded, respectively, for M agalactiae and MmmLC.

Inactivation of Mycoplasma species involved in contagious agalactia.

The suitability of 5 agents for the inactivation of different field strains of the four mycoplasma species associated with contagious agalactia syndrome in goats, i.e. Mycoplasma agalactiae, Mycoplasma mycoides subsp. mycoides LC, Mycoplasma capricolum subsp. capricolum and Mycoplasma putrefaciens, was investigated. Immunoprophylaxis of this syndrome is still based on inactivated vaccines, which traditionally use formalin as the inactivating agent. Moreover, the limited information existing about this type of vaccine is only based on assays against Mycoplasma agalactiae. Our results showed that formalin (0.1%, 37 degrees C during 16 hours) and phenol (0.5%, 24 hours) were effective against all species tested. Surprisingly, binary ethileneimine (BEI), a classical virus-inactivating agent, also proved to be very effective when it was used in a 0.1 M concentration over 24 hours. With heat treatment, every species was inactivated at 60 degrees C. No satisfying results were obtained with purified saponin. To evaluate the harmful effects of each agent on mycoplasmal proteins, a representative strain was subjected to an effective inactivation protocol with each agent, which was monitored by Western immunoblotting. Immunoblotting was performed using sera of animals inoculated with the respective mycoplasma species, to compare the effect of all the agents on treated strains with untreated strains. The results confirmed that phenol, BEI and to a lesser extent also formalin inactivated all species without causing a significant damage while heat caused stronger damage on surface proteins. Future in vivo studies should be conducted because, as recently shown, the combined use of a suitable inactivant and adjuvant could give rise to the induction of certain cytokines and strong antibody production of a specific isotype pattern, thus opening ways to develop more efficacious inactivated vaccines against contagious agalactia.

A specific PCR for the detection of Mycoplasma putrefaciens, one of the agents of the contagious agalactia syndrome of goats.

Mycoplasma putrefaciens is listed as one of the etiologic agents of the contagious agalactia syndrome by the world organisation for animal health. This species has been characterized only recently, 1974, and the number of outbreaks caused by this microorganism so far is very scarce. It induces mastitis in infected goats although other symptoms such as arthritis in adults and septicaemia in kids are also frequently described. Up to now, the identification of M. putrefaciens relied on classical isolation and identification techniques which present a number of limitations. Specific primers for PCR have been designed based on sequence comparisons of the ArcB gene among the 'Mycoplasma mycoides cluster' and related species such as Mycoplasma cottewii and Mycoplasma yeatsii. Sequence alignments confirmed the taxonomic position of M. putrefaciens, which is related to the 'M. mycoides cluster' but also very close to M. yeatsii. The polymorphism observed amongst the different ArcB sequences allowed the determination of a primer pair yielding a specific amplification of a 316 bp-long DNA fragment by PCR. This PCR was validated in two different laboratories with a variety of mycoplasma strains isolated from goats. This new PCR technique will be very useful for a quicker determination of M. putrefaciens strains as well as a better understanding of the prevalence of M. putrefaciens infections.

[The role of staphylococci in the occurrence, development and chronicity of lactation mastitis]. / Rol' stafilokokkov v vozniknovenii, razvitii i khronizatsii laktatsionnykh mastitov.

The investigation results obtained by the authors and the data of literature on the role of the biological properties of staphylococci in the pathogenesis, development and chronization of acute suppurative lactation mastitis are generalized. The concept of lactation mastitis as a monoetiological disease caused solely by bacteria of the genus Staphylococcus is substantiated. The importance of the individual properties of staphylococci at different stages of the pathogenesis of lactation mastitis, as well as their role in ensuring the resistance of the infective agent to the protective mechanisms of the body and in the chronization of the suppurative process, is considered. The data on the use of a number of biological properties of staphylococci as markers for the prognostication of the course of lactation mastitis are presented and their prognosticating effect at different tactics of surgical treatment is evaluated.

Selective acylation of plasma membrane proteins of Mycoplasma agalactiae: the causal agent of agalactia.

Revealed by in vivo labeling with (14)C-palmitic acid, about 15 acylated proteins were identified in the plasma membrane of Mycoplasma agalactiae (type strain PG2), including the major component p40. Triton X-114 phase partitioning and Western blotting demonstrated the amphiphilic properties of the acyl proteins and showed that they were also antigenic components. Chemical analyses of fatty acids bound to proteins revealed the following selectivity order within acylation: stearic acid (18:0) > linoleic acid (18:2c) approximately palmitic acid (16:0) > oleic acid (18:1c) > myristic acid (14:0), with 16:0 and 18:1c preferred for the O-acylation and 18:0 for the N-acylation. The ratio [O-ester- + amide-bound acyl chains]/O-ester-linked chains being close to 1.4 as well as the presence of S-glycerylcysteine suggest that acyl proteins in M. agalactiae are true lipoproteins containing N-acyl diacyl glycerylcysteine, probably processed by a mechanism analogous to that described for Gram-negative eubacteria.