Experimental bovine genital ureaplasmosis. I. Granular vulvitis following vulvar inoculation.

Granular vulvitis was reproduced in ten virgin heifers following vulvar inoculation with strains of ureaplasma previously isolated from natural cases. The disease appeared one to three days postinoculation and was characterized by vulvar swabs but not from the upper mucopurulent discharge. At necropsy 13 to 41 days later, ureaplasmas were recovered consistently from vulvar swabs but not from the upper reproductive tract. It was concluded that some strains of ureaplasma are pathogenic and should be viewed as a cause of bovine granular vulvitis.     

Experimental Bovine Genital Ureaplasmosis II. Granular Vulvitis, Endometritis and Salpingitis Following Uterine Inoculation

Twenty-three virgin Holstein heifers received uterine inoculations with ureaplasma and were necropsied one to thirteen days later. Three heifers inoculated intracervically were necropsied on days 3, 5 and 11.

Granular vulvitis was produced on average by 3.6 days in fourteen of sixteen uterine inoculated heifers monitored for four or more days. Two cervically inoculated heifers monitored for over four days also developed granular vulvitis by the fourth day.

At necropsy, ureaplasma was recovered from 94% of uterine horn cultures for the first four days postinoculation and 50% during days 5 to 7. Thereafter all uterine cultures were negative. The percentage of positive ureaplasma recoveries from uterine tube flushings was lower than for uterine horns but remained positive for a longer period. By day 7, three of four uterine tube flushings were still positive. No bacterial pathogens were isolated from the uterine horns or uterine tube flushings.

On histopathology 50% of uterine inoculated heifers had endometritis up to six days postinoculation and a slightly higher percentage (58%) had salpingitis. Endometritis was not found in any heifers after day 6. Residual salpingitis was present in one heifer on day 7. Endometritis was present in cervically inoculated heifers necropsied on days 3 and 5 but not on day 11. Salpingitis was not found in any of the three cervically inoculated animals.

The study concluded that some strains of ureaplasma are pathogenic for the upper reproductive tract of the cow and should be considered significant when isolated from cases of granular vulvitis, endometritis or salpingitis.

     

Isolation of Ureaplasma from bovine granular vulvitis.

Cultures for mycoplasmatales, viruses and bacteria were made from bovine vulvar swabs to determine whether ureaplasma was associated with a clinical granular vulvitis observed in 16 Ontario dairy herds. Ureaplasma was isolated from 23.5% of 34 clinically normal cows, 74% of 27 cows with mild to moderate vulvar hyperemia but no discharge and 100% of 20 cows with acute vulvar hyperemia accompanied by purulent discharge. There were statistically significant differences in rates of isolation among clinical groups. Mycoplasma bovigenitalium was isolated from 7.7% and 20% of cows with moderate or acute vulvitis respectively but not from normal cows. Haemophilus somnus was isolated from 25% of cows with acute vulvitis. There were no significant differences in isolations of Escherichia coli, Corynebacterium pyogenes and alpha-hemolytic streptococcus between normal and clinically affected animals. Cultures of 135 repeat samples from 33 cows revealed that ureaplasma persisted in some animals for at least three months. No viruses were isolated from any of the animals in this study.     

In vitro antimicrobial susceptibility of Mycoplasma mycoides mycoides large colony and Arcanobacterium pyogenes isolated from clinical cases of ulcerative balanitis and vulvitis in Dorper sheep in South Africa

The in vitro activities of enrofloxacin, florfenicol, oxytetracycline and spiramycin were determined against field isolates of Mycoplasma mycoides mycoides large colony (MmmLC) by means of the broth microdilution technique. The minimum inhibitory concentrations (MICs) of these antimicrobial drugs were determined for a representative number of 10 isolates and 1 type strain. The susceptibility of Arcanobacterium pyogenes to enrofloxacin, oxytetracycline and tilmicosin was determined by means of an agar disk diffusion test. The MICs of enrofloxacin, florfenicol, oxytetracycline and spiramycin were within the ranges of 0.125-0.5, 1.0-2.0, 2.0-4.0 and 4.0-8.0 microg/ml, respectively. This study has shown that resistance of MmmLC against enrofloxacin, florfenicol, oxytetracycline and spiramycin was negligible. All the field strains of A. pyogenes that were tested were susceptible to enrofloxacin, oxytetracycline and tilmicosin with mean inhibition zones of 30.6, 42.3 and 35.8 mm, respectively. Although there is lack of data on in vivo efficacy and in vitro MIC or inhibition zone diameter breakpoints of these antimicrobial drugs for MmmLC, the MIC results indicate that these 4 classes of antimicrobial drugs should be effective in the treatment of ulcerative balanitis and vulvitis in sheep in South Africa.     

Insertion sequence profiling of UK Mycoplasma bovis field isolates

Mycoplasma equigenitalium and Mycoplasma subdolum have been associated with infertility, endometritis, vulvitis and abortions in mares, and with reduced fertility and balanoposthitis in stallions. Despite their role in equine genital disorder, determinants of virulence and pathogenesis as well as factors provoking specific host immune responses have not been identified, so far. To establish the major immunogenic components of Mycoplasma (M.) equigenitalium and M. subdolum, antigen profiles of their type strains as well as 30 clinical isolates were compared by SDS-PAGE and immunoblot analysis using hyperimmune rabbit sera and equine sera from clinical cases. To define the major protein antigens of both mycoplasma species, detergent-phase fractionation with Triton X-114 was performed. Western blot analysis of 30 clinical isolates revealed a high similarity of their overall antigen profile with only slight differences. In contrast, monospecific polyclonal antibodies raised against detergent-phase proteins of the two mycoplasma species identified three prominent proteins (pST17, pST42, and pET45) undergoing variation in expression and size among clinical and clonal isolates.     

Bovine Genital Mycoplasmosis

Infection, lesions and clinical significance of Acheloplasmas, Mycoplasma bovis and Mycoplasma bovigenitalium in genital disease of cattle are described. A more detailed account is given of ureaplasma infections. Acute and chronic forms of granular vulvitis in both field and experimental disease are described as well as the role of the organism in abortion.

Recovery rates of ureaplasma and mycoplasma from semen and preputial washings in bulls are outlined and their significance in disease is discussed. There are problems in differentiating pathogenic from nonpathogenic isolates. Methods are being developed to treat semen for these organisms.

This paper provides a concise summary of clinical and microbiological aspects of bovine genital mycoplasmosis.

     

Ulcerative vulvitis and balanitis in sheep flocks

Outbreaks of ulcerative vulvitis and balanitis occurred in three commercial sheep flocks in England and Wales. Between 29 and 44 per cent of the ewes were affected; most of the lesions resolved in three weeks. Pathogens such as mycoplasmas, which have previously been associated with these conditions, were not detected despite using improved laboratory techniques. In one of the flocks, ovine herpesvirus type 2 was detected by pcr in the blood of two acutely affected ewes, from the vulval ulcers of one of them, and from the penis of an affected ram.     

Isolation of Ureaplasma diversum and mycoplasmas from genital tracts of beef and dairy cattle in Saskatchewan

We report herein a survey in which cultures of bovine reproductive tracts for Ureaplasma diversum and mycoplasmas were carried out in order to better understand the role of these organisms in granular vulvitis (GV). Samples cultured were vulvar swabs from clinically normal cows or ones with GV, preputial swabs or raw semen from bulls, and abomasal contents of aborted fetuses.

Ureaplasma diversum was isolated from 104 (43.3%) of 240 dairy cows, 32 (27.1%) of 118 beef cows, 43 (47.2%) of 91 beef heifers, 23 (67.6%) of 34 beef bulls, and three (60%) of five dairy bulls. Mycoplasmas were isolated from 18 (7.5%) dairy cows, two (1.6%) beef cows, three (8.8%) beef bulls, and one dairy bull. No isolation was made from 97 aborted fetuses. For 65 dairy cows and 30 beef heifers with vulvar lesions, the isolation rates for ureaplasmas of 62.5% and 69.7%, respectively, were significantly higher (X²) than those for normal animals (37.5% and 30.3%). On immunofluorescent serotyping of 137 of the 205 isolates, there were 66 in serogroup C (strain T44), 18 in serogroup B (strain D48), eight in serogroup A (strain A417 or strain 2312), 14 cross-reacting, and 31 that were not identified. It was concluded that U. diversum is commonly present in the lower reproductive tract of beef/dairy cattle in Saskatchewan and is associated with granular vulvitis.

     

Identification and Molecular Characteristics Analysis of Porcine Epidemic Diarrhea Virus Variants

To ascertain a diarrhea case in a pig farm in Shandong province,the pathological changes of dead piglets were observed and nested RT-PCR test was carried out on 7 diarrhea samples for porcine epidemic diarrhea virus (PEDV).Pathological examination revealed that intestine was detected as enlargement,hyperemia and edema.After histological examination,the typical microscopic lesions of intestine were disappearance of epithelial cells,villus shrinkage and shortening.The result of nested RT-PCR showed that all of the 7 samples could amplify a specific target band of PEDV.Molecular characteristics of two field strains showed that they had an amino acid homology of 92.3% to 92.4% with vaccine CV777 and 96.6% to 98.6% with other previous field strains which sequences were downloaded from GenBank.Phylogenetic tree analysis further revealed that all of PEDV strains could be mainly divided into two clusters of G1 and G2. G2 consisted of the field strains of our study and other field strains from USA,China and so on,which had the same sequence characteristics of two insertions and one deletion,while G1 consisted of all vaccines of CV777 and several older field strains from China and Korea.These results indicated that our field strains were the dominant strains in recent epidemic diarrhea occurrence.Moreover,its molecular characteristics might be a characterization of differentiating the field and vaccine strains.     

猪流行性腹泻病毒变异株的鉴定和分子特征分析

为查明山东省某猪场流行性腹泻的病因,本研究对该猪场病死仔猪进行病理学检查,并对采集的7份仔猪腹泻样品进行猪流行性腹泻病毒(PEDV)的套式RT-PCR检测。病理学检查发现病猪的肠道肿胀、变薄、出血,肠绒毛皱缩、变短、边缘粗糙。套式RT-PCR结果显示,7份样品均为PEDV阳性;对其中两份PEDV阳性病料进行S基因扩增,分析其遗传变异,结果显示这两株PEDV野毒株的S基因与参考疫苗株CV777的同源性为92.3%~92.4%,与其他野毒株的同源性为96.6%~98.6%。PEDV的遗传进化树结果显示,PEDV分为两个进化分支G1和G2,G1包括CV777等疫苗株及中国和韩国早期的部分毒株,G2则是PEDV变异株为主的分支,包括本试验分离的野毒株及近年来美国、中国等国的野毒株,这些毒株都存在两个插入突变和1个缺失突变。结果表明,变异株已成为PEDV的流行优势毒株,且这些变异位点有可能成为鉴定PEDV变异株的分子特征。     

Use of restriction fragment length polymorphism, immunoblotting, and polymerase chain reaction in the differentiation of avian poxviruses.

Restriction deoxyribonucleic acid (DNA) fragment profile analysis coupled with immunogenic protein profile analysis has provided useful information in determining the differences between vaccine strains and field isolates of fowlpox virus (FPV). The DNA of strains examined in this study clearly fell into 3 minor groups of restriction patterns similar but distinct from one another: restriction patterns exhibited by the vaccine strains except 1 vaccine strain, Vac-82; restriction profiles indicated by Vac-82 and field isolates FI-38 and FI-42; and restriction patterns indicated by field isolates FI-43, FI-51, FI-54, and FI-56. Furthermore, when the strains were analyzed and compared by immunoblotting analysis, they showed group differences similar to the differences in restriction profiles. Both techniques provided high sensitivity in verifying differences between vaccine strains and field isolates of FPV. The disparity found in restriction fragments or immunogenic protein profile between vaccine strains and field isolates does not exclude the appreciable high degree of DNA sequence conservation and homology. However, the minor disparity observed in these strains suggests a molecular basis for why vaccinated commercial flocks could have continually been infected by variant strains of FPV. A rapid and sensitive polymerase chain reaction method, which amplified a product from the 4b core protein gene of the FPV genome, was developed for identification and differentiation of members of the genus Avipoxvirus. Whereas total DNA from either vaccine strains or field isolates was used as template for amplifying a predicted product of 578 or 1409 bp, only cleavage of the amplified product (1409 bp) represented an additional detection technique for species differentiation. An attempt to distinguish between strains on the basis of amplification product was partially successful.     

Molecular analysis of field strains of Mycoplasma capricolum subspecies capripneumoniae and Mycoplasma mycoides subspecies mycoides, small colony type isolated from goats in Tanzania.

A molecular analysis of strains of Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae) and Mycoplasma mycoides subsp. mycoides, small colony type (M. mycoides SC) isolated from goats was performed using the amplified fragment length polymorphism (AFLP) and pulsed-field gel electrophoresis (PFGE) fingerprinting techniques. Among the 11 field strains of M. capripneumoniae from Tanzanian goats, two AFLP patterns were demonstrated, with 10 of the strains showing indistinguishable patterns. Five Kenyan strains of M. capripneumoniae produced three AFLP patterns, with two of them being indistinguishable from the 10 identical Tanzanian and one Ugandan strain (M74/93) isolated from sheep. The AFLP pattern of the type strain (F38(T)) was identical to two Kenyan strains (Baringo and G183/82). On PFGE analysis, all the examined M. capripneumoniae strains exhibited identical PFGE profiles.Five field strains of M. mycoides SC isolated from goats displayed identical AFLP patterns except for one strain which differed from others at only one position. The AFLP pattern of the type strain of M. mycoides SC (PG1(T)) was different from the field strains. The five field strains of M. mycoides SC produced identical PFGE profiles, which were, however, different from the type strain. The AFLP and PFGE profiles of M. mycoides SC strains from goats were identical to those of six strains isolated from cattle affected with contagious bovine pleuropneumonia (CBPP) in the same areas. The results of this study suggest a close epidemiological linkage between strains of M. capripneumoniae and between M. mycoides SC type, respectively, isolated from goats in Tanzania.     

Molecular phylogenetic analysis of bovine papular stomatitis viruses detected in Saga,Japan

In this study, we performed a molecular phylogenetic analysis of six bovine papular stomatitis virus (BPSV) field strains detected from Japanese beef calves kept on a farm in Saga prefecture, a southwest part of Japan, from 2017 to 2020. The phylogenetic analysis based on a partial B2L gene (554-nt) showed that these field strains were divided into two lineages, a lineage (A-lineage) constructed by a Saga strain and strains obtained from various regions of Japan and the world, and other lineage (B-lineage) constructed by five Saga strains and strains obtained from France, USA and Iwate prefecture (a north part of Japan). Furthermore, a Saga field strain named BPSV_SAGAbv2 and strains obtained from USA and Iwate prefecture belonged to a sub-lineage blanched from B-lineage. This is the first report elucidating molecular epidemiological characters of field BPSVs obtained from Saga prefecture. The existence of the multiple lineages was thought to be related to a history of calf introduction from various regions of Japan into Saga prefecture.     

Monoclonal antibody characterization of two field strains of Haemophilus paragallinarum isolated from vaccinated layer hens

An oil-based bacterin, containing strains 083 and 0222 of Haemophilus paragallinarum, is commonly used in South Africa to vaccinate laying flocks against infectious coryza. Two strains of H. paragallinarum, designated M85 and SB86, were isolated from infected but vaccinated commercial laying flocks in two incidental outbreaks of coryza in 1985 and 1986. A panel of five monoclonal antibodies was established which clearly distinguished the vaccine strains from the field isolates. One of these reacted with only vaccine strains A and B, another reacted with only field strains M85 and SB86, and the remaining three cross-reacted to various degrees with all four strains or isolates. Immunoassays were performed by enzyme-linked immunosorbent assay using whole bacteria as solid-phase antigen. These monoclonal antibodies may aid in serotyping new field isolates of H. paragallinarum and in improved standardization of vaccine strains.     

Molecular epidemiological investigation of foot-and-mouth disease virus in Korea in 2000

The genetic relatedness of 7 Korean type O field strains of foot-and-mouth disease virus (FMDV) in clinical specimens collected from 5 different geographic locations in 2000 was investigated. The sequence of 162 nucleotides (nt 478-639) at the 3' end of the 1D (VP1) genes was determined from amplified cDNA fragments, and subjected to the analysis for the sequence identity/divergence and phylogenetic relationship. The overall nucleotide sequence divergence among the 7 field strains was 0 to 3.8%, suggesting that they are closely related to each other. Phylogenetic analysis with the known Middle East-South Asia (ME-SA) topotype strains showed that the 7 Korean field strains formed two distinct clusters within the same lineage of the ME-SA topotype strains. Cluster 1 consisted of the strains of the primary foci of infection (Paju and Hongseong), and closely related to the strains prevailed in the Far East. Cluster 2 comprised those of subsequently affected regions (Boryeong, Yongin, and Chungju), and was further diverged from the Cluster 1. The result of phylogenetic analysis indicated that the Korean strains may have evolved from a common ancestor of the Pan Asia strains, and that at least 2 phylogenetically clustered variants within the same lineage were prevalent during the epidemic. The potential origin and sources of the virus introduction to Korea were discussed.     

DNA fingerprinting for differentiation of field isolates from reference vaccine strains of Pasteurella multocida in turkeys

The genomes from field isolates of Pasteurella multocida in turkeys and those of P multocida reference CU and M9 vaccine strains were analyzed and compared after cleavage with restriction endonucleases. The electrophoretic profiles obtained with DNA fragments from field isolates and vaccine strains of the same serotype were characteristic and reproducible. These features indicated the existence of differences among the isolates of the same serotype that cannot currently be detected, using available serotyping methods. However, several field isolates had electrophoretic profiles similar to those of either CU or M9 vaccine strain. It was concluded that restriction endonuclease analysis of DNA genomes from P multocida isolated from turkeys provides the information for differentiation of field isolates from vaccine strains of the same serotype.     

Detection of Ureaplasma diversum in cattle using a newly developed PCR-based detection assay

Ureaplasma diversum has been associated with different clinical manifestations including bovine vulvitis, endometritis, salpingitis, spontaneous abortion and infertility. Because the isolation of this ureaplasma from clinical samples is difficult, there is a need for improved detection methods. We developed a PCR assay based on amplification of a region of the gene encoding 16S rRNA. The specificity of the amplification was verified by sequence analysis. Female bovine vaginal swabs (n=168) were collected and the presence of U. diversum evaluated by both culture methods and by the PCR assay. Culture was positive for 60 samples (35.7%), and PCR-specific amplification was obtained for 89 samples (52.9%). These results indicated a high prevalence of U. diversum in the selected animals and the higher sensitivity of this PCR assay as compared to culture.     

我国部分地区猪瘟病毒流行株的基因差异

采用 RT-PCR方法 ,从我国 1 0个省、市、自治区收集的 2 3个猪瘟病毒 ( CSFV)流行毒株中 ,扩增了CSFV E2基因中主要抗原位点编码区。对各扩增片段进行了序列测定 ,通过计算机分析构建了系统发生树 ,并确定了它们间的遗传相关性。结果表明 ,2 3个流行毒株中的 1 8株属基因 群 ,占 78.2 6%;另外 5个流行株与传统石门强毒、兔化弱毒株属基因 群 ,占 2 1 .74 %。两群间测序区的核酸同源性只有 78.90 %。此外 ,根据序列差异程度 ,将基因 群流行株分为 3个亚群 ,各基因群 CSFV在地域分布上未发现有明显的特征性。本研究初步揭示了我国较大范围内流行的 CSFV毒株与传统的石门强毒和疫苗用兔化弱毒在抗原基因上存在较大的差异 ,以及我国猪瘟病毒流行株在地域分布上的多样性