Presence of contagious agalactia causing mycoplasmas in Spanish goat artificial insemination centres

Male goats admitted to artificial insemination centres come from herds that have shown no clinical symptoms of contagious agalactia (CA) for the last 6 mo. However, prior reports suggest that this control measure may not be completely effective. This study was designed to detect the presence of CA-causing mycoplasmas in 9 Spanish centres, comprising 159 goats (147 males and 12 teaser does) of 8 different breeds. A microbiological study was conducted during 8 mo on 448 samples (318 ear swabs, 119 semen samples and 11 milk samples). In 86 samples (84 swabs, 1 semen sample and 1 milk sample), CA-causative mycoplasmas were detected by PCR or culture, and 52 animals (49 goat males and 3 teaser does) tested positive. Most of these positive animals were auricular carriers (n = 50), mainly of Mycoplasma mycoides subsp. capri (Mmc), although some M. agalactiae (Ma) and, interestingly, M. capricolum subsp. capricolum (Mcc) carriers were also identified. At least 1 animal infected by CA-causing mycoplasmas was detected in 8 of the 9 centres (88.8%) although in most (66.7%) no infected animals or only 1 or 2 positive animals were identified. Our results indicate the presence of CA carriers as asymptomatic animals in reproductive programmes. These findings have already prompted efficient measures to detect and avoid the entry of these carriers in Spanish centres. We recommend similar measures for all centres in areas where CA is endemic.     

Mycoplasma agalactiae detected in the semen of goat bucks

Contagious agalactia (CA) is among the most significant diseases affecting small ruminant populations in Mediterranean countries. This study was designed to detect the excretion in semen of CA-causing mycoplasmas in goats (Capra hircus) reared in Spain, where the disease is considered endemic. Culture techniques and PCR were conducted on 147 semen samples collected from 113 goat bucks to detect mycoplasmas. No animal showed clinical symptoms of CA at the moment of the screening. M. agalactiae was identified using both diagnostic methods in three semen samples collected from three different bucks. These animals belonged to a group of animals in which semen had been analyzed twice and only the second sample proved positive, suggesting the possibility of intermittent excretion. This is the first report of the isolation of M. agalactiae from semen collected from naturally infected goats. Future studies should investigate whether semen could be a real source of CA infection by determining if the agent may be transmitted during natural service or when semen is used for artificial insemination.     

Prevalence of mycoplasmas in external ear canal of goats: Influence of the sanitary status of the herd

A study was carried out in 15 French goat herds to assess the presence of mycoplasmas in the external ear canal of goats and to estimate the association between the ear isolates (highly pathogenic or non-pathogenic) and the status of the herds with respect to the mycoplasma infections. In each herd, 20 clinically normal goats were investigated once and mycoplasma isolates were identified by MF-dot tests. Influence of herd status on the proportion of animals carrying highly pathogenic species of mycoplasmas was evaluated by calculating the Odds Ratios. The present study gave first of all an estimate of the relative importance of mycoplasma carriage in healthy French dairy goat herds. The species isolated were MmmLC, Mcc, Ma, Mp (considered as highly pathogenic) and My and Mc (non-pathogenic species). Carriage often involve several strains and/or species in the same ear canal. The highly pathogenic species were significantly more frequent in the herds with prior history of mycoplasma infection. This asymptomatic carrier state makes the sanitary status of a herd ambiguous and represents a risk of subsequent propagation of the etiological agents, during close physical contact or by intervention of vectors (mites).     

Surveillance of Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri in dairy goat herds

This study was designed to monitor the presence of Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri (Mmc) in 66 dairy goat herds of a genetic improvement programme in a region of Spain where contagious agalactia is endemic. Over a whole lactation period, 300 bulk tank milk and 381 milk samples from goats with clinical mastitis were subjected to polymerase chain reaction (PCR) to detect the two mycoplasma species. The presence of mycoplasmas (either species or both) was detected in 66.7% of the herds and M. agalactiae was identified in 95.45% of these positives herds. In a given infected herd, mycoplasmas were not continuously detected over the whole study period. Our findings indicate that in an endemic area, M. agalactiae and Mmc can be monitored through PCR analysis of mastitic milk and bulk tank milk (BTM) samples. Over a lactation period we recommend testing multiple BTM samples on a herd. No relationship was observed between the use of inactivated mycoplasma vaccines and the PCR detection of both mycoplasmas.     

Mycoplasmas Isolated from the Respiratory Tract of Cattle and Goats in Tanzania

A microbiological study of the mycoplasma flora in the respiratory tracts of cattle and goats in selected regions of Tanzania is described. In the examination of cattle, mycoplasmas were isolated from 60 (17.8%) of the 338 examined lung samples, 8 (47.1%) of the 17 lymph nodes, 4 (13.3%) of the 30 pleural fluid samples and 4 (3.9%) of the 103 nasal swabs examined. All the isolates were identified as Mycoplasma mycoides subsp. mycoides, Small Colony type except for one isolate from pleural fluid which was identified as Mycoplasma arginini. M. mycoides subsp. mycoides, Small Colony type was isolated from samples originating from Dodoma, Iringa, Mbeya, Morogoro and Shinyanga regions where outbreaks of contagious bovine pleuropneumonia had been reported. In the examination of goats, mycoplasmas were isolated from 54 (34.0%) of the 159 examined lung samples, 41 (18.1%) of the 226 nasal swabs and 4 (40.0%) of the 10 pleural fluid samples. The species demonstrated were Mycoplasma capricolum subsp. capripneumoniae, M. mycoides subsp. mycoides, Small Colony type Mycoplasma ovipneumoniae and M. Capricolum subsp. arginini. The isolation of M. capripneumoniae in the Coast and Morogoro regions confirmed the presence of contagious caprine pleuropneumonia in the regions.     

Real-Time PCR Investigation of Potential Vectors, Reservoirs, and Shedding Patterns of Feline Hemotropic Mycoplasmas

Three hemotropic mycoplasmas have been identified in pet cats: Mycoplasma haemofelis, “Candidatus Mycoplasma haemominutum,” and “Candidatus Mycoplasma turicensis.” The way in which these agents are transmitted is largely unknown. Thus, this study aimed to investigate fleas, ticks, and rodents as well as saliva and feces from infected cats for the presence of hemotropic mycoplasmas, to gain insight into potential transmission routes for these agents. DNA was extracted from arthropods and from rodent blood or tissue samples from Switzerland and from salivary and fecal swabs from two experimentally infected and six naturally infected cats. All samples were analyzed with real-time PCR, and some positive samples were confirmed by sequencing. Feline hemotropic mycoplasmas were detected in cat fleas and in a few Ixodes sp. and Rhipicephalus sp. ticks collected from animals but not in ticks collected from vegetation or from rodent samples, although the latter were frequently Mycoplasma coccoides PCR positive. When shedding patterns of feline hemotropic mycoplasmas were investigated, “Ca. Mycoplasma turicensis” DNA was detected in saliva and feces at the early but not at the late phase of infection. M. haemofelis and “Ca. Mycoplasma haemominutum” DNA was not amplified from saliva and feces of naturally infected cats, despite high hemotropic mycoplasma blood loads. Our results suggest that besides an ostensibly indirect transmission by fleas, direct transmission through saliva and feces at the early phase of infection could play a role in the epizootiology of feline hemotropic mycoplasmas. Neither the investigated tick nor the rodent population seems to represent a major reservoir for feline hemotropic mycoplasmas in Switzerland.     

Real-time PCR investigation of potential vectors, reservoirs, and shedding patterns of feline hemotropic mycoplasmas

Three hemotropic mycoplasmas have been identified in pet cats: Mycoplasma haemofelis, "Candidatus Mycoplasma haemominutum," and "Candidatus Mycoplasma turicensis." The way in which these agents are transmitted is largely unknown. Thus, this study aimed to investigate fleas, ticks, and rodents as well as saliva and feces from infected cats for the presence of hemotropic mycoplasmas, to gain insight into potential transmission routes for these agents. DNA was extracted from arthropods and from rodent blood or tissue samples from Switzerland and from salivary and fecal swabs from two experimentally infected and six naturally infected cats. All samples were analyzed with real-time PCR, and some positive samples were confirmed by sequencing. Feline hemotropic mycoplasmas were detected in cat fleas and in a few Ixodes sp. and Rhipicephalus sp. ticks collected from animals but not in ticks collected from vegetation or from rodent samples, although the latter were frequently Mycoplasma coccoides PCR positive. When shedding patterns of feline hemotropic mycoplasmas were investigated, "Ca. Mycoplasma turicensis" DNA was detected in saliva and feces at the early but not at the late phase of infection. M. haemofelis and "Ca. Mycoplasma haemominutum" DNA was not amplified from saliva and feces of naturally infected cats, despite high hemotropic mycoplasma blood loads. Our results suggest that besides an ostensibly indirect transmission by fleas, direct transmission through saliva and feces at the early phase of infection could play a role in the epizootiology of feline hemotropic mycoplasmas. Neither the investigated tick nor the rodent population seems to represent a major reservoir for feline hemotropic mycoplasmas in Switzerland.     

Caprine mycoplasmal mastitis in Nigeria

In a study to investigate the current status of intramammary mycoplasmosis in caprine udders in Nigeria, a total of 57 and 24 milk samples were collected from udders of goats affected by mastitis and from apparently normal goats’ udders, respectively. Acute and chronic mastitis were more commonly observed in goats between 1 and 3 years old. Mycoplasma agalactiae and Mycoplasma capricolum occurred at a significantly higher rate (p<0.05) in udders affected by mastitis than in normal healthy udders. Other mycoplasma occurring in low prevalence include Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides LC. It is concluded that cultural (microbiological) surveillance is necessary for effective treatment and control of the disease in Nigeria.     

T-strain mycoplasma in the chimpanzee.

Specimens from 4 chimpanzees were cultured for T-strain mycoplasma and Mycoplasma hominis. T-strain mycoplasmas were recovered from the genital tract and throat of a male and the genital tract of his female cagemate; neither had clinical evidence of infection. Two other male chimpanzees were culturally negative for T-strain mycoplasmas. M hominis was not isolated from any of the animals. The chimpanzee may serve as a suitable experimental model for studying the role of T-strain mycoplasmas in human urethritis and reproductive failure.     

PCR和分离培养同时检测儿童呼吸道感染的支原体

为了解广州市儿童呼吸道支原体感染情况,用一条共同的上游引物,二条特异性的下游引物建立的ＰＣＲ方法能同时扩增ＭＰ的６９１ｂｐ和ＭＧ的４３８ｂｐ粘附因子基因片段,但不会扩增其他支原体和细菌的ＤＮＡ。     

Further evidence to justify reassignment of Mycoplasma mycoides subspecies mycoides Large Colony type to Mycoplasma mycoides subspecies capri

Analysis, using the polymerase chain reaction (PCR), restriction enzyme endonuclease analysis (REA), protein profile patterns, random amplification of polymorphic DNA (RAPD) fingerprinting, 16S rRNA gene sequencing and antisera growth inhibition tests, of 22 strains of Mycoplasma mycoides subsp. mycoides Large Colony type (MmmLC) and eight strains of M. mycoides subsp. capri (Mmc) are presented, along with a summary of comparative data from the literature for over 100 strains, all of which supports the reclassification of the MmmLC and Mmc strains into the single subspecies, M. mycoides subspecies capri.     

Fertility prediction in dairy goats from Murciano-Granadina breed: The role of sperm evaluation and female traits

Fertility is one of the most economically important traits in farm animals, due to the direct and indirect costs associated to low pregnancy rates. Thus, one of the priority goals in animal reproduction is to predict the performance that the semen doses will have in vivo based on the quality values obtained in laboratory assays. Attempts have been made for getting a predictive model of fertility of frozen-thawed sperm in dairy goats, but similar studies have not been conducted for chilled goat buck sperm doses that are mostly used for artificial insemination in many countries including Spain. We study how parameters of in vitro sperm quality and characteristics of Murciano-Granadina dairy goats may affect the in vivo fertility obtained after artificial insemination with semen doses chilled at 4 °C. Moreover, this information was used for obtaining predictive models of the fertility. Sixty-three ejaculates from 13 males were used to prepare chilled doses for the insemination of 495 goats over 13 sessions. Fresh and chilled sperm were evaluated for motility and plasma membrane integrity with a computer-assisted sperm analysis system and flow cytometry, respectively. Fertility was determined at parturition, according to the kidding goats. Overall fertility was 59.6%. Pearson’s correlation coefficients between in vivo fertility and quality variables of fresh sperm were not significant and were low (below 0.34 in absolute value) for chilled sperm. Females’ characteristics had a low negative impact on fertility (correlation coefficients of ?0.19 with age, ?0.20 with parturitions and ?0.11 with total milk yield obtained in the best lactation). Fixed and mixed logistic regression procedures were used trying to explain the fertility results. None of the models accurately predicted fertility, but the best models included the percentage of total motile sperm or average path velocity from fresh semen, age of the females and the session effect (uncontrolled environmental effects). These analyses showed that primiparous goats were 2.42 times more likely to get pregnant than goats that had kidded four or more times. Our field assay data on fertility in Murciano-Granadina dairy goats highlighted the importance of making quality controls of sperm, of choosing the doses presenting high percentages of motile sperm exhibiting regular trajectories and of selecting the youngest goats for AI, after their first kidding. Efforts should continue to obtain better predictive models for improving fertility in goat dairy herds.     

咽拭子快速培养在肺炎支原体感染中的临床应用价值

目的:探讨咽拭子快速培养在肺炎支原体感染中的临床应用价值。方法:收集2014年2月~2016年2月期间我院收治的呼吸道感染患儿220例,用肺炎支原体专用液体培养基进行肺炎支原体快速培养,用胶体金法检测肺炎支原体MP-Ig M。比较两种方法的阳性率。结果:咽拭子培养快速培养阳性率与血清MP-Ig M检测阳性率比较,差异无统计学意义(P0.05)。MP-Ig检测显示,≤1岁阳性率最低,其阳性率随年龄增加不断增高(P0.05)。肺炎支原体咽拭子培养显示,≤1岁阳性率最高,2~8岁最低(P0.05)。病程≤7 d患者肺炎支原体咽拭子培养阳性率(34.21%)显著高于肺炎支原体MP-Ig检测阳性率(14.04%)(P0.05)。病程7 d患者肺炎支原体咽拭子培养阳性率(11.32%)显著低于肺炎支原体MP-Ig检测阳性率(52.83%)(P0.05)。肺炎支原体咽拭子培养的灵敏度性以及特异性显著高于肺炎支原体MP-Ig检测,差异具有统计学意义(P0.05)。结论:咽拭子快速培养对肺炎支原体感染的早期诊断有一定临床应用价值,方法简单,无创伤,值得临床进一步研究和应用。     

Detection of Leptospira spp. in semen and vaginal fluids of goats and sheep by polymerase chain reaction

Thirteen goat herds and seven sheep flocks in the state of Rio de Janeiro, Brazil were screened for leptospirosis. From the three herds and three flocks with greatest seroreactivity, 19 goats (16 females and three bucks) and 40 sheep (26 ewes and 14 rams) that were seropositive (specific anti-Leptospira titres > or =400, based on a microscopic agglutination test), were selected for more detailed studies. From those animals, samples of vaginal fluids or semen were collected for bacteriological and molecular assays. For both species of animals, the most prevalent reactions were to serovars Hardjo, Shermani, and Grippotyphosa. Although leptospires were detected by darkfield microscopy in three vaginal fluid samples (from two goats and one ewe), pure isolates were not obtained by bacteriological culture of vaginal fluids or semen. However, seven vaginal fluid samples (from four goats and three ewes) and six semen samples (all from rams) were positive on polymerase chain reaction (PCR). Based on these findings, in addition to analogous findings in cattle, we inferred that there is potential for venereal transmission of leptospirosis in small ruminants.     

Effect of different diluents on goat semen fertility

Skim milk (SM) is considered to be the most widely employed extender for goat sperm used for artificial insemination (AI). However, the fertilizing life span of sperm stored in milk or milk-based extenders does not exceed 12h. Besides some seminal plasma components, such as a protein fraction from the goat bulbourethral gland secretion (SBUIII), interacts with some milk fractions and inhibits the spermatozoa motility. The aim of this study was to prolong the survival of buck semen and its fertility. Buck ejaculates were diluted to a final concentration of 100x10(6)spermatozoa/ml with three different diluents: SM, TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) and TEMPOL+hyaluronic acid (TEMPOL+HA). At 7h from dilution 42 goats were inseminated with semen diluted with SM (short-term semen) while after storage for 24h, 44 and 45 goats were inseminated with semen diluted with TEMPOL and TEMPOL+HA (long-term storage), respectively. At day 50 from AI the percentages of pregnant goats were 71.4% (30/42) with SM, 61.4% (27/44) with TEMPOL and 48.8% (22/45) with TEMPOL+HA, with significant differences between SM and TEMPOL+HA. The kidding rate was 66.7% (28/42) with SM diluent, 61.4% (27/44) with TEMPOL and 48.8% (22/45) with TEMPOL+HA, without significant differences among treatment groups. In conclusion, it is possible to maintain good fertility in goats after AI with semen stored for 24h in TEMPOL.     

Social rank and response to the "male effect" in the Australian Cashmere goat

The present study was conducted to determine if the social status of Australian Cashmere goats affects their response to the male effect in terms of LH secretion, ovulation and expression of estrus. Australian Cashmere goats were kept isolated from the males during 5 months. The index of success (SI) of each goat was calculated to establish their social rank. In the first experiment, the ten most dominant and the 10 most subordinate goats were separated from the original herd and housed in two pens (5 dominant and 5 subordinate animals in each pen). An androgenized wether was then introduced into each pen. Luteinizing hormone (LH) was measured every 20 min from 2h before to 4h after introduction of the male in the goats of first pen and from 4 to 8h after male introduction in the second pen. In the second experiment, the remaining 50 goats were exposed in their original pen to two androgenized wethers. Their association index with the males (AI) was calculated for each of these 50 goats, and the intervals from exposure to the males to the onset of estrus and to ovulation were determined. During the first 4h after male introduction, the dominant goats had more LH pulses (0.65+/-0.06 compared with 0.3+/-0.09; P<0.05) and greater LH mean concentrations (1.79+/-0.14 ng/ml compared with 1.30+/-0.15 ng/ml, P=0.05) than the subordinate animals. Although not significantly different, the AI was 35% greater for high and medium ranking goats than for low ranking animals (0.031+/-0.004, 0.032+/-0.005 and 0.023+/-0.005, respectively, P>0.05). Although the number of goats ovulating in response to male exposure was similar between dominance groups (high: 100%, medium: 94% and low ranking: 92%), the high and medium dominance goats showed a greater incidence of expression of estrus than low-dominance goats (94.4%, 89.5% and 53.8%, respectively, P<0.05). It is concluded that the social rank of the Australian Cashmere goat influences their response to the male effect in terms of early LH secretion and expression of estrus.     

Enriching the captive elephant population genetic pool through artificial insemination with frozen-thawed semen collected in the wild

The first successful AI in an elephant was reported in 1998, using fresh semen. Since then almost 40 calves have been produced through AI in both Asian and African elephants worldwide. Following these successes, with the objective of enriching the captive population with genetic material from the wild, we evaluated the possibility of using frozen-thawed semen collected from wild bulls for AI in captivity. Semen, collected from a 36-yr-old wild African savanna elephant (Loxodonta africana) in South Africa was frozen using the directional freezing technique. This frozen-thawed semen was used for four inseminations over two consecutive days, two before and two after ovulation, in a 26-yr-old female African savanna elephant in Austria. Insemination dose of 1200 × 10⁶ cells per AI with 61% motility resulted in pregnancy, which was confirmed through ultrasound examination 75, 110 and 141 days after the AI procedure. This represents the first successful AI using wild bull frozen-thawed semen in elephants. The incorporation of AI with frozen-thawed semen into the assisted reproduction toolbox opens the way to preserve and transport semen between distant individuals in captivity or, as was done in this study, between wild and captive populations, without the need to transport stressed or potentially disease-carrying animals or to remove animals from the wild. In addition, cryopreserved spermatozoa, in combination with AI, are useful methods to extend the reproductive lifespan of individuals beyond their biological lifespan and an important tool for genetic diversity management and phenotype selection in these endangered mammals.     

Infectious keratoconjunctivitis in bighorn sheep, Silver Bell Mountains, Arizona, USA

An infectious keratoconjunctivitis (IKC) epizootic in bighorn sheep (Ovis canadensis) occurred in the Silver Bell Mountains, Arizona, USA, from 1 December 2003 to 31 March 2004. We used standard culture methods and polymerase chain reaction (PCR) amplification of the 16S rRNA gene to test for the causative agents of IKC and other diseases reported to be associated with bighorn sheep populations. All bighorn sheep and domestic goat test results were negative except for Mycoplasma spp. and Branhamella spp. The culture and PCR results differed. Conjunctival swabs from four of 19 IKC-affected bighorn sheep tested by culture were positive for Mycoplasma spp., whereas 22 of 22 bighorn sheep samples tested by PCR were positive for Mycoplasma spp. None of 13 domestic goats tested positive by culture for Mycoplasma spp., whereas five of 16 tested positive by PCR. Three of 16 domestic goats and seven of 24 IKC-affected bighorn sheep tested positive for Branhamella spp. by culture. Bighorn sheep began showing clinical signs of IKC between 21 and 28 days following initial detection of domestic goats in bighorn sheep habitat. The IKC epizootic lasted 122 days, and individual bighorn sheep were blind for an average of 38.4 days. Given the clear potential for disease transmission to bighorn sheep, we recommend that land managers not allow the pasturing of domestic goats near occupied bighorn sheep habitat.     

Effect of Mycoplasma bovis and Mycoplasma bovigenitalium in semen on fertilization and association with in vitro produced morula and blastocyst stage embryos

Frozen-thawed bovine semen contaminated with Mycoplasma bovis (M. bovis) or Mycoplasma bovigenitalium (M. bovigenitalium) at either a high (10(6) CFU/mL) or low (10(4) CFU/mL) concentration was used for bovine oocyte insemination. The resulting embryos were washed 10 times as recommended by the International Embryo Transfer Society (IETS) prior to isolation of agent. A total of 1494 oocytes was inseminated with contaminated sperm cells and 855 oocytes with uninfected control semen. There was a significantly higher proportion of embryos that developed to the blastocyst stage in control than in the mycoplasma exposed groups (P<0.05). Isolation of motile spermatozoa by swim-up procedure prior to insemination did not render sperm cells free of Mycoplasma spp. Although M. bovis was isolated from all washed embryos after the high exposure level, it was found in only 60% of the samples after the low exposure level. In contrast, M. bovigenitalium was isolated from 70 and 12% of washed embryos exposed to the high and low levels of microorganism, respectively. Using scanning electron microscopy, both microorganisms were detected in association with the surface of zona pellucida-intact embryos and with sperm cells. These results indicate that mycoplasmas present in semen can be transmitted through the IVF system and infect embryos. Furthermore, the experiments showed that supplementation of culture media with standard antibiotics and washing embryos as recommended by IETS were not effective in rendering IVF embryos free from M. bovis and M. bovigenitalium.     

Detection and identification of Mycoplasma infections by DNA hybridization

Infection of cell cultures by mycoplasmas can be detected by hybridization of the DNA of suspected cell cultures with recombinant plasmids containing fragments of the mycoplasma DNA. The test is very sensitive and allows detection of as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA amount of 10(6) mycoplasmas. This approach turns out to be effective for detection and identification of mycoplasmas in clinical material, plant and insect tissues. A set of DNA probes for detection of mycoplasmas infecting cell cultures by dot hybridization has been constructed. This set consists of specific DNA probes and universal DNA probe. Recombinant plasmids, pAl32, pMa13, pMh9, containing specific DNA fragments of Acholeplasma-laidlawii, Mycoplasma arginini, Mycoplasma hominis (the prevalent mycoplasma contaminants of home cell cultures) are species-specific DNA probes. Recombinant plasmid pMg16 containing rRNA genes of Mycoplasma gallisepticum is the universal DNA probe for detection of any mycoplasma (or any prokaryote) contaminations. These two classes of DNA probes may be considered as complementing each other. These 32P labeled probes do not hybridize with eukaryotic DNA. The set of DNA probes allows not only to detect infection of cell cultures by mycoplasmas but also to identify the species of mycoplasmas and to evaluate the multiplicity of mycoplasma infection.