Prevalence of Potentially Pathogenic Bacteria as Genital Pathogens in Dairy Cattle

Bacteria on the genital mucosa have been studied less in healthy, non-puerperal cows than in cows with puerperal endometritis. We have thus analysed bacteria in swabs from the vagina and cervix of post-puerperal cattle (n = 644). Out of the animals, 6.8% had aborted within the last 12 months, 2.6% and 11.6% showed signs of vaginitis and endometritis, respectively. In 17.2% of cervical swabs pathogenic gram-positive and in 11.5% pathogenic gram-negative bacteria were found. Arcanobacterium pyogenes was isolated from 41.3% of cows with endometritis and from 3.5% without endometritis (p < 0.05). From 12.5% of cows with abortion but from no cow without abortion, Staphylococcus aureus was recovered (p < 0.05). Out of 383 vaginal swabs, 88.3% were positive. In 3.4% of swabs pathogenic gram-positive and in 16.7% pathogenic gram-negative microorganisms were found. The percentage of positive vaginal swabs did not differ between pregnant and non-pregnant animals. In the genital tract, the percentage of swabs positive for normal mucosal bacteria decreased from caudally to cranially (p < 0.05). Pathogenic bacteria were found more often in cervical than in vaginal swabs (p < 0.05). In conclusion, bacteria on the vaginal and cervical mucosa in cattle involve a wide range of species. In animals without endometritis or vaginitis, colonization of the mucosa rather than infection has to be assumed.     

Investigation of Chlamydophila spp. in dairy cows with reproductive disorders

Background

Reports worldwide indicate high prevalence of Chlamydophila spp. infection in cattle. To assess the prevalence in Sweden, 525 cows in 70 dairy herds with reproductive disorders was investigated.

Methods

To detect antibodies two commercially available kits were used. Moreover, 107 specimens, including vaginal swabs, organ tissues and milk were analysed by Polymerase Chain Reaction (PCR).

Results

Two (0.4%) cows were seropositive in the Pourquier Cp. abortus ELISA. The seroprevalence with the Chekit ELISA was 28% with no difference between cases and controls. Five specimens were positive in real-time PCR and further analysed by nested PCR. Cp. pecorum was confirmed by partial omp1 DNA sequencing of the nested PCR product of vaginal swabs from control cows.

Conclusion

The results suggest that Cp. abortus infection is absent or rare in Swedish cows whereas Cp. pecorum is probably more spread. They also suggest that Chlamydophila spp. are not related to reproduction disorders in Swedish cattle.     

Examination of cattle with respiratory diseases for Mycoplasma and bacterial bronchopneumonia agents

A total of 247 mycoplasma strains was isolated from 435 lungs, tracheobronchial secretions and nasal swabs originating from cattle with symptoms of bronchopneumonia. Mycoplasma (M.) bovis was found 89 times (36%) and was the most common mycoplasma species in the lungs. M. bovirhinis, M. bovigenitalium, M. spec. and Acholeplasma (A.) laidlawii were isolated 158 times (64%). Among these mycoplasmas M. bovirhinis was the most widespread species (114 isolations). In 55 cases (62%) M. bovis was associated with Pasteurella or Actinomyces (A.) pyogenes. The other mycoplasma species were found in 67 cases (42%) together with these bacteria. Without mycoplasmas Pasteurella and A. pyogenes occurred in 33 of the probes investigated (21%). Beside mycoplasmas Haemophilus (H.) somnus was isolated from 16 of 162 tracheobronchial secretions investigated. The results confirm earlier suppositions that in most of the cases bronchopneumonia of cattle is a multifactorial event, frequently associated with mycoplasmas--especially M. bovis.     

Prevalence of Chlamydophila abortus infection in domesticated ruminants in Taiwan.

This study is to (1) investigate the prevalence of Chlamydophila abortus infection in cows and goats in Taiwan, and (2) compare the genetic properties of Taiwanese isolates with abortion strains from other sources. Approximately 71% of aborted cows and 58% of aborted does had IgG against C. abortus in their sera. The seroprevalence rate in cows may be overestimated, because a certain degree of cross-reactivity with C. pecorum cannot be ruled out. Only 22.7% (from aborted cows) and 33.3% (from aborted dogs) of vaginal swabs that tested positive by polymerase chain reaction led to successful isolation of C. abortus by inoculation into chicken embryos, equivalent to 7.1% and 7.9% of isolation rates, respectively. The major outer membrane protein gene of 15 Taiwanese abortion isolates was compared with that of various strains by restriction fragment length polymorphism (RFLP) and nucleotide sequencing. Restriction enzyme CfoI was able to distinguish Taiwanese ruminant isolates, which have identical RFLP patterns, from C. felis (feline) and C. psittaci (avian) strains. Taiwanese isolates had 98.8-100% homology with known ruminant abortion strains and were phylogenetically closest to bovine LW508 strain.     

A field study of Mycoplasma bovis infection in cattle

After an outbreak of mastitis in cattle caused by Mycoplasma bovis a study was made in 5 herds with recent cases (principal herds) and in 4 control herds. In the principal herds, M. bovis was isolated from milk samples, nasal swabs, and from one vaginal swab. M. bovis was also isolated from nasal swabs of calves in 2 of the 4 control herds, whereas all milk samples and vaginal swabs from the control herds were negative. Evaluation of serum antibody titres to M. bovis among non-mastitic animals of 3 principal herds and 1 control herd showed no difference in distribution of the titre values, which generally were low. However, cows excreting M. bovis in the milk had high antibody titres. The way of introduction to the herds and the spread of the infection within the herds could not be established by the study, which was supplemented by a DNA restriction fragment analysis of a number of M. bovis isolates.     

Chlamydiaceae infections in pig

ABSTRACT: Chlamydiaceae are Gram-negative obligate intracellular bacteria. They are responsible for a broad range of diseases in animals and humans. In pigs, Chlamydia suis, Chlamydia abortus, Chlamydia pecorum and Chlamydia psittaci have been isolated. Chlamydiaceae infections in pigs are associated with different pathologies such as conjunctivitis, pneumonia, pericarditis, polyarthritis, polyserositis, pseudo-membranous or necrotizing enteritis, periparturient dysgalactiae syndrome, vaginal discharge, return to oestrus, abortion, mummification, delivery of weak piglets, increased perinatal and neonatal mortality and inferior semen quality, orchitis, epididymitis and urethritis in boars. However, Chlamydiaceae are still considered as non-important pathogens because reports of porcine chlamydiosis are rare. Furthermore, Chlamydiaceae infections are often unnoticed because tests for Chlamydiaceae are not routinely performed in all veterinary diagnostic laboratories and Chlamydiaceae are often found in association with other pathogens, which are sometimes more easily to detect. However, recent studies have demonstrated that Chlamydiaceae infections in breeding sows, boars and piglets occur more often than thought and are economically important. This paper presents an overview on: the taxonomy of Chlamydiaceae occurring in pigs, diagnostic considerations, epidemiology and pathology of infections with Chlamydiaceae in pigs, public health significance and finally on prevention and treatment of Chlamydiaceae infections in pigs.     

Identification and characterisation of coding tandem repeat variants in incA gene of Chlamydophila pecorum

Bacteria of the family Chlamydiaceae are obligate intracellular pathogens of human and animals. Chlamydophila pecorum is associated with different pathological conditions in ruminants, swine and koala. To characterize a coding tandem repeat (CTR) identified at the 3' end of incA gene of C. pecorum, 51 strains of different chlamydial species were examined. The CTR were observed in 18 of 18 tested C. pecorum isolates including symptomatic and asymptomatic animals from diverse geographical origins. The CTR were also found in two strains of C. abortus respectively isolated from faeces from a healthy ewe and from a goat belonging to asymptomatic herds, but were absent in C. abortus strains isolated from clinical disease specimens, and in tested strains of C. psittaci, C. caviae, C. felis and C. trachomatis. The number of CTR repeats is variable and encode several motifs that are rich in alanine and proline. The CTR-derived variable structure of incA, which encode the Chlamydiaceae-specific type III secreted inclusion membrane protein, IncA, may be involved in the adaptation of C. pecorum to its environment by allowing it to persist in the host cell.     

The indirect hemagglutination test for the detection of antibodies in cattle naturally infected mycoplasmas.

Stable mycoplasma antigens for the indirect hemagglutination test (IHA) were prepared employing glutaraldehyde treated sheep erythrocytes sensitized with Mycoplasma agalactiae subsp. bovis and Mycoplasma bovigenitalium antigens. Employing these antigens mycoplasma antibodies were detected in sera from cattle which had mastitic symptoms due to natural infection with either M. agalactiae subsp. bovis or M. bovigenitalium. A total of 200 cows from four herds were examined at varying intervals for the presence of M. agalactiae subsp. bovis and for the detection of antibody using growth inhibition and IHA tests. Mycoplasmas were isolated from 37 animals. Growth inhibiting antibody was detected from 56 of the 200 animals. In the IHA tests, antibody titer greater than or equal to 1:80 were detected in 148 animals, 76 of these having antibody titers greater than or equal to 1:160, while sera of 116 normal control animals had no growth inhibiting antibody and none had IHA antibody titers greater than 1:40. M. bovigenitalium was isolated from the milk of three of 26 animals in a fifth herd during an outbreak of mastitis. Growth inhibiting antibodies were demonstrated in the sera of ten of the 26 animals. However, the IHA test detected antibody titers of greater than or equal to 1:160 in 13 animals and of 1:80 in one of the 26 animals. To determine the specificity of the IHA tests, M. agalactiae subsp. bovis and M. bovigenitalium antigens were reacted with rabbit hyperimmune typing sera produced against 12 species of bovine mycoplasmatales. Homologous antisera showed IHA antibody titers of 1:1280 and 1:2560 against M. agalactiae subsp. bovis and M. bovigenitalium respectively, whereas heterologous antisera showed IHA antibody titers of less than or equal to 1:20. Also eight type-specific bovine antisera were reacted with M agalactiae subsp. bovis and M. bovigenitalium antigens in homologous and heterologous tests. Homoogous reactions showed IHA antibody titers greater than or equal to 1:320, whereas heterologous reactions showed IHA titers of less than or equal to 1:20. This IHA test promises to be useful for the detection of bovine mycoplasma antibodies in sera from cattle infected with M. agalactiae subsp. bovis or M. bovigenitalium. Thes test is sensitive, reproducible and specific and the technique is relatively simple and rapid. The antigens were stable for at least seven months.     

Seroprevalence of chlamydial infection in cattle in Ireland

Although few studies have investigated the prevalence of chlamydial infections in cattle, reported prevalence rates vary hugely. In order to assess the prevalence of this infection in cattle in Ireland, serum samples (100 herds, 20 samples/herd) collected for statutory screening for brucellosis were examined by soluble chlamydial antigen indirect ELISA. The assay detects antibodies to the two most common Chlamydiaceae spp. affecting cattle, namely Chlamydia abortus and Chlamydia pecorum. A total of 95 samples from 57 herds were seropositive, representing an observed prevalence rate of 4.75%. The parametric bootstrap estimate of the mean disease prevalence in the population was 6.04% (95%, CI 4.70-7.50). The results suggest the prevalence of chlamydial infection is low in cattle in Ireland.     

Phenotypic characterization of Brucella strains isolated from livestock in Nigeria

Isolation of brucellae from aborted fetuses, hygroma fluids, milk and vaginal swabs obtained from aborting cattle, sheep, goats, pigs, and horses in Nigeria was carried out. A total of 25 isolates, obtained mainly from cattle, sheep and horses, were biotyped. All strains belonged to one species, Brucella abortus biovar 1. The epidemiological significance of this finding is discussed. Some preliminary observations on the zoonotic and public health implications of Brucella infection in Nigerian livestock are presented. A control programme involving improved management, animal movement restrictions, public health education and mass vaccination of animals is suggested.     

Diagnostic investigation into the role of Chlamydiae in cases of increased rates of return to oestrus in pigs

Cervical swabs and serum samples were taken from Swiss herds of sows with high rates of irregular return to oestrus (group A) and from control herds without reproductive problems (group B. The genital tracts of 21 slaughtered sows of group A were also examined. The swabs and genital tracts were screened for Chlamydiae by a new 16S rRNA PCR and the sera by an ELISA for Chlamydiaceae lipopolysaccharide. Chlamydophila (Cp) abortus was isolated from seven of the 65 swabs taken from group A but from none of the 128 swabs taken from group B. Chlamydia suis was present in swabs from both groups A (1.5 per cent) and B (2.3 per cent). In addition, Cp abortus was detected in 33.3 per cent of the genital tracts. Of the 193 sera tested, 61.7 per cent were positive, with no significant difference between group A (52.3 per cent) and group B (66.4 per cent). Chlamydia-like organisms were detected in 28.2 per cent of the swabs from group A and in 22 per cent of those from group B.     

Efficacy of live Chlamydophila abortus vaccine 1B in protecting mice placentas and foetuses against strains of Chlamydophila pecorum isolated from cases of abortion

The efficacy of Chlamydophila abortus vaccine strain 1B in protecting against two selected Chlamydophila pecorum strains, isolated from an aborted goat (M14) in Morocco and a ewe (AB10) in France, was investigated in a mouse model, by comparing the reduction in number of bacteria in the placentas of vaccinated mice challenged intraperitoneally at 11 days of pregnancy with the reference C. abortus (AB7) and C. pecorum (M14, or AB10) strains, to those of unvaccinated mice. Vaccine 1B was shown to provide effective protection against the field strains of C. pecorum, since it significantly reduced the placental Chlamydophila colonisation. The two C. pecorum strains were not sufficiently abortifacient in mice to use reduction in abortion as a criterion of protection.     

Molecular evidence for chlamydial infections in the eyes of sheep

Ocular infections by chlamydiae are associated with ocular disease manifestations such as conjunctivitis and keratitis in humans and animals. Limited evidence exists that members of the order Chlamydiales can also cause ocular disease in sheep. In the current study, the prevalence of chlamydiae in the eyes of sheep was investigated by using PCR methods. Data obtained in sheep by broad-range 16S rRNA order Chlamydiales-specific PCR were compared to the prevalence of antibodies against chlamydiae detected by a competitive enzyme-linked immunosorbent assay (cELISA). Flocks tested included a clinically healthy flock and two flocks suffering from ocular disease and with histories of Ovine Enzootic Abortion (OEA). PCR detected DNA of Chlamydophila (Cp.) abortus and Cp. pecorum in the eyes of both healthy and sick animals but also identified Chlamydia (C.) suis and a variety of uncultured chlamydia-like organisms. Good correlation was found between the presence of Cp. abortus DNA in sheep conjunctival samples and seropositivity detected by cELISA. Despite these findings, no association was found between the presence of chlamydial DNA in the sheep conjunctival samples and the onset of clinical disease. These results suggest that the biodiversity of chlamydiae in the eyes of sheep is greater than that previously thought. Further investigations are needed to determine whether a causal relationship between infection by chlamydiae and ocular disease exists in these animals.     

Detection of mycoplasmas in experimentally-infected bull sperm

Bull sperm pellets experimentally infected with various concentrations of Mycoplasma (M.) bovis, M. bovigenitalium, and M. californicum were stored several months at -196 degrees C and were subsequently tested for mycoplasmas, using different methods and culturing media. M. bovis was reliably identified by laboratory diagnosis up to a concentration of 10(2) cfu/pellet. M. bovigenitalium and M. californicum were found to make higher demands on the quality of culturing substrates. Recommended are 1-h broth incubation and 5-d broth breeding.     

Waddlia, Parachlamydia and Chlamydiaceae in bovine abortion

The etiology remains unknown in many cases of bovine abortion in Switzerland. Bacteria of the Chlamydiales order are known abortive agents, therefore cases of bovine abortion from three representative regions of Switzerland were investigated in this study. Particularly Chlamydiaceae as well as the Chlamydia-like organisms Waddlia and Parachlamydia were of interest, especially because of their possible zoonotic potential. Placenta samples (n=343) were tested for these bacteria by different PCR-methods, immunohistochemistry and serology for Chlamydia abortus. Additionally an attempt for the isolation of Waddlia and Parachlamydia was made by co-cultivation in amoebae. In 67.3% of the 343 cases a necrotizing and/or purulent placentitis was found histologically. By real-time PCR, 0.9% (3/343) of the cases were positive for Waddlia, 13.4% (46/343) positive for Parachlamydia and 14.6% (50/343) positive or questionable positive for Chlamydiaceae. Of these samples, confirmation by immunohistochemistry was possible in 2/3 cases for Waddlia, 25/46 for Parachlamydia and 4/50 for Chlamydiaceae. Of the 50 cases positive or questionable positive for Chlamydiaceae, species-identification by ArrayTube Microarray or 16S rRNA PCR resulted in 41 cases positive for C. abortus whereas the presence of Chlamydia suis was confirmed in four and Chlamydia pecorum in one case. This study brought evidence for the importance of different members of Chlamydiales in different regions of Switzerland although Waddlia is not occurring in a high prevalence. On the other hand mixed infections with different Chlamydiales as well as with other abortigenic agents could be found.     

Mixed infections in vitro with different Chlamydiaceae strains and a cell culture adapted porcine epidemic diarrhea virus

Assuming a synergistic or additive effect of Chlamydiaceae in coexistence with other enteropathogenic agents, the viral/bacterial interaction between a cell culture adapted porcine epidemic diarrhea virus (ca-PEDV) and different Chlamydiaceae strains was studied in vitro. Vero cells were dually infected with ca-PEDV and one of the three chlamydial strains Chlamydia trachomatis S45, Chlamydophila abortus S26/3 or Chlamydophila pecorum 1710S. Three experimental protocols were designed varying the inoculation sequence. Cell layers were first inoculated with Chlamydiaceae and 20 h later with ca-PEDV in protocol one. In protocol two, both agents were administered concurrently, whereas in protocol three, ca-PEDV was applied 20 h in advance of the Chlamydiaceae. Immunofluorescence techniques, immunohistochemical (IH) staining and electron microscopy were subsequently employed to investigate the cell layers. Using indirect immunofluorescence (IF) labeling, all mixed infections revealed dually infected cells, however, only incidentally and in low numbers. Characteristically, ca-PEDV syncytia with one or more chlamydial inclusions were detected but dually infected single cells were absent. Some syncytial cells contained enlarged C. abortus or C. pecorum inclusions with abnormally large developmental forms. In comparison with simultaneously conducted monoinfections, larger chlamydial inclusions were observed in dually infected cell layers. Experiments with C. trachomatis showed significantly increased numbers of chlamydial inclusions in dually infected cell layers compared to monoinfected ones. These findings indicate an influence of ca-PEDV on the chlamydial developmental cycle and in the case of C. trachomatis, a positive effect on chlamydial colonization in mixed infections.     

Use of a polymerase chain reaction for detection of Mycoplasma dispar in the nasal mucus of calves.

A polymerase chain reaction (PCR) assay was developed for the detection of Mycoplasma dispar in nasal mucus samples collected from calves. The target DNA sequence was the 16S rRNA gene, and the fragment was selected within a region of high polymorphism. The specificity and detection limit of the method were determined. This method was then used for the detection of M. dispar in nasal swabs collected from 301 calves, including 155 clinical samples from animals showing signs of respiratory disease and 146 samples from healthy animals. PCR with generic primers was applied to the detection of Mollicutes, followed by the detection of M. dispar. Mollicutes were detected in 52.05% of clinical samples from healthy animals and in 90.96% of samples from sick animals. Mycoplasma dispar was detected in 6.16% of healthy animals and in 34.84% of sick animals. The PCR assay was useful in verifying the presence of M. dispar in calves and may be a useful tool in monitoring this mycoplasma in cattle herds.     

Mycoplasma species and related organisms isolated from ruminants in Britain between 1990 and 2000

Between 1990 and 2000, more than 1600 mycoplasmas and the related acholeplasmas were identified from ruminant animals by the Mycoplasma Group at the Veterinary Laboratories Agency--Weybridge. Mycoplasma bovis was the most commonly identified pathogen, mostly from pneumonic calves but occasionally from cattle with mastitis and arthritis. Mycoplasma canis was first isolated in Britain in 1995 from pneumonic calves and the number of isolates increased to 18 per cent of the total mycoplasmas isolated from cattle in 1999. The ELISA for antibodies to M. bovis detected 1971 positive samples (22 per cent) among 8959 serum samples, mainly from pneumonic cattle. Other mycoplasmas identified included Mycoplasma dispar from the lungs of cattle with respiratory disease, and Mycoplasma bovigenitalium from the reproductive tract of cows with vulvovaginitis and infertility. Mycoplasma bovirhinis and Acholeplasma species were found commonly but are thought to be more opportunistic than pathogenic. In sheep and goats, the majority of Mycoplasma species isolated were identified as Mycoplasma ovipneumoniae from pneumonic sheep, Mycoplasma conjunctivae from sheep with keratoconjunctivitis, and the ubiquitous Mycoplasma arginini.     

Associations between the prevalence of Mollicutes and Mycoplasma bovis and health and performance in stocker calves

A longitudinal cross-sectional time-series study was carried out to determine the prevalence of nasal mycoplasma carriage, serostatus and seroconversion, and to evaluate the associations between these parameters and health and performance in weaned beef calves during a 42-day feeding period. Nasal swabs and serum were collected on days 0 (arrival), 10, 42 and at the first incidence of bovine respiratory disease complex. The samples were evaluated for Mollicutes (by culture), Mycoplasma bovis (by PCR) and serum antibody to M. bovis. On day 0, 90.4 per cent of the calves were Mollicutes nasal culture-positive. The seroprevalence of M. bovis was 26.6 per cent on day 0 and 98.2 per cent by day 42 (P<0.05). Seroconversion to M. bovis between days 0 and 42 was significantly associated (P=0.04) with lower weight gain. Weight gain was greater in calves that were PCR-negative for M. bovis on day 10 (P=0.01). The percentage of calves seropositive to M. bovis increased throughout the study, indicating exposure and an immunological response to the organism. Although associations with health outcomes were not identified, seroconversion to M. bovis was associated with a decreased rate of weight gain during the study period.