Abortion In Ruminants Research Articles

Isolation of listeria monocytogenes in neural forms of listeriosis and abortions in ruminants

L. monocytogenes is a food borne pathogen capable of causing serious invasive diseases in humans and animals, including abortion, septicemia, meningitis and meningoencephalitis. Isolation of the agent is the most accurate diagnostic method in all situations of suspected L. monocytogenes infection. Direct isolation of L. monocytogenes is relatively simple in cases when the number of organisms is very large, such as septicaemic disease forms. Isolation is quite difficult if the agent is present in very small quantities, such as in encephalitis, or if the sample is highly contaminated. In this paper we presented the isolation of L. monocytogenes from clinical samples by using selective media for enrichment and isolation, combined with the cold enrichment technique. In our investigation we isolated L. monocytogenes from 18 of the total of 46 investigated tissue samples originating from animals with clinical diagnosis of listeriosis. We also presented the basic differential-diagnostic procedure in relation to the mimicking bacterial species.

Development and production of vaccines against abortion caused by Chlamydophila abortus and Coxiella burnetii in small ruminants

Chlamydophila abortus and Coxiella burnetii are obligate intracellular parasites, small Gram-negative bacteria, which are aetiological agents of abortion in small ruminants. The organisms also have zoonotic potential, which highlights the importance of the prevention of the transmission of these diseases. Treatment of infected animals with oxytetracycline and its derivatives, can limit abortions, but does not suppress the bacterial excretion, therefore the only effective solution against the infection is the prevention by vaccination. To produce and make available such vaccines, is a long process from the emergence of a concept, through feasibility, development, production, control until obtaining marketing authorisation, and it can take 5–10 years or more. After registration, pharmacovigilance follows the vaccines on the field. C. burnetii and Cp. abortus present serious health risk to humans (Class II and III micro-organisms), thus the propagation of living bacteria is performed in confined Class III areas. Our vaccine strain against chlamydiosis is Cp. abortus 1B ts, whilst the strain of vaccine developed against coxiellosis is C. burnetii Nine Mile phase I. We present hereby the advantages of these vaccines produced with the strains mentioned above over other, existing vaccines and describe their major phases of production.

Analysis of the Behavior of eryC Mutants of Brucella suis Attenuated in Macrophages

The facultatively intracellular pathogen Brucella, characterized by its capacity to replicate in professional and non professional phagocytes, also causes abortion in ruminants. This property has been linked to the presence of erythritol in the placenta, as brucellae preferentially utilize erythritol. The ery operon encodes enzymes involved in erythritol metabolism, and a link with virulence has since been discussed. Allelic exchange mutants in eryC of Brucella suis were erythritol sensitive in vitro with a MIC of 1 to 5 mM of erythritol. Their multiplication in macrophage-like cells was 50- to 90-fold reduced, but complementation of the mutant restored wild-type levels of intracellular multiplication and the capacity to use erythritol as a sole carbon source. In vivo, the eryC mutant colonized the spleens of infected BALB/c mice to a significantly lower extent than the wild type and the complemented strain. Interestingly, eryC mutants that were in addition spontaneously erythritol tolerant nevertheless exhibited wild-type-like intramacrophagic and intramurine replication. We concluded from our results that erythritol was not an essential carbon source for the pathogen in the macrophage host cell but that the inactivation of the eryC gene significantly reduced the intramacrophagic and intramurine fitness of B. suis.

Influence of the Th2 immune response established by Nippostrongylus brasiliensis infection on the protection offered by different vaccines against Chlamydophila abortus infection

Chlamydophila abortus is the aetiological agent of enzootic abortion in small ruminants in which it infects the placenta to cause abortion during the last trimester of gestation. In a mouse model, a Th1 immune response involving IFN-gamma production and CD8+ T cells is necessary for the infection to be resolved. The authors previously demonstrated that infection with Nippostrongylus brasiliensis, a rodent gastrointestinal nematode extensively used in experimental models to induce Th2 responses, alters the specific immune response against C. abortus infection, increasing bacterial multiplication in liver and reducing specific IFN-gamma production. The aim of the present work was to clarify whether a Th2 immune response has any influence on the success of vaccination using both inactivated and attenuated vaccines. The results showed that the Th2 response established prior to vaccination did not influence the induction of protection offered by the vaccines. However, the effectiveness of this protective response can be altered, depending on the adjuvant employed in the inactivated vaccines, when the Th2 response is established after vaccination, just before challenge with C. abortus.

Loss of Milk Yield due to Akabane Disease in Dairy Cows

Akabane disease is an infection with clinical signs of congenital malformation and abortion in ruminants. Abnormal parturitions caused by Akabane disease result in great economic loss. The purpose of this study is to estimate the reduction in the milk yield from abnormal parturition due to Akabane disease. The data were collected from 33 Holstein cows on 11 farms. The animals had abnormal parturitions during the period from September 1998 to March 1999, and were diagnosed as having Akabane disease. The mean and standard deviation of the rate of reduction in the milk yield of 33 cows after abnormal parturition caused by Akabane disease was -11.4 +/- 14.9%. The means and standard deviations of the rate of reduction of four cows calving abnormally at 220-239 days of gestation, nine cows calving abnormally at 240-270 days of gestation, and 20 cows calving abnormally at 271-300 days of gestation were -26.6 +/- 24.7%, -14.7 +/- 11.0%, and -6.9 +/- 12.3%, respectively. In this study, we demonstrated that the rate of reduction in the milk yield in cows affected by Akabane disease was -11.4 +/- 14.9%, but values as high as -26.6 +/- 24.7% were reached in the comparison with the milk yield obtained after normal parturition.

Evaluation of ovine abortion associated with Toxoplasma gondii in Spain by different diagnostic techniques

A total of 173 aborted ovine foetuses and seven aborted caprine foetuses, submitted from different points of north and central Spain, were analysed to determine the role of T. gondii in abortion and to compare the utility of the most widely used techniques in diagnosis of the congenital infection (histopathology, serology—IFAT and ELISA—and a nested-PCR). Parasite infection was diagnosed in 40 (23.1%; n=173) ovine foetuses by at least one of the diagnostic techniques used. A higher percentage of foetuses were diagnosed using serological techniques (IFAT and ELISA) (28.3%; n=106) than by histologic examination (8.7%; n=173) or PCR (6.9%; n=173). No significant association between infection and the foetal age categories was found ( P>0.05). In this study, 106 aborted foetuses were analysed by all of the three diagnostic techniques. When we compared serological results, perfect agreement between ELISA and IFAT was obtained. On the contrary, slight to fair agreements were observed when histology results were compared with those obtained by serology and PCR techniques. All the positive foetuses were aborted in the mid (60%) or last (40%) term of pregnancy, but no significant differences were found between ages of the infected and non-infected foetuses ( P>0.05). This report indicates that toxoplasmosis may be a common cause of small ruminant abortion and neonatal death in Spain and points out the necessity of using different and complementary techniques to increase the probability of detecting Toxoplasma infection in an aborted foetus.

Application of touchdown enzyme time release (TETR)-PCR for diagnosis of Chlamydophila abortus infection

Chlamydophila abortus-DNA was detected using a touchdown enzyme time-release (TETR)-polymerase chain reaction (PCR) assay as an improved test for sensitive and rapid diagnosis of abortion in small ruminants. Two hundred and fifty two placentae, liver or spleen tissue samples from aborting ewes and goats or aborted lambs and kids in which C. abortus infection was suspected were examined by TETR-PCR and the results were compared with cell culture. Sixty-five tissue samples were found to be TETR-PCR positive while only 56 samples were cell culture-positive. After resolution of discrepant samples with a confirmatory nested PCR assay, TETR-PCR had a sensitivity of 97% and a specificity of 99.5% while culture had a sensitivity of 84.8% and a specificity of 100%. The analytical sensitivity of the TETR-PCR assay was determined with DNA extracted from 4-fold serial dilution of C. abortus B577 culture and found to be 0.25 inclusion-forming unit per PCR. No reduction in the analytical sensitivity was noted when the assay was tested with mouse liver samples spiked with 4-fold serial dilution of C. abortus B577 culture. No target product was amplified when DNA from Chlamydophila pecorum was tested. TETR-PCR used in this study is a practical, rapid, sensitive and specific assay that could be used for the detection of C. abortus in infected tissue samples. We recommend the use of this assay as a supplemental diagnostic tool for detection of C. abortus in infected tissue samples.

Abortion in small ruminants in Switzerland: investigations during two lambing seasons (1996-1998) with special regard to chlamydial abortions

Abortion cases of 144 goats und 86 sheep were investigated etiologically during 2 lambing seasons (1996/1997, 1997/1998). Macroscopic inspection of fetus and placenta was completed by histopathology and bacteriological isolation of agents. In addition, immunohistologically the following antigens were labeled in formalin-fixed and paraffin-embedded tissue sections: Toxoplasma gondii, Neospora caninum, Chlamydophila abortus (formerly Chlamydia psittaci serovar 1) and Border Disease Virus. From farms with abortions caused by Chlamydophila abortus specific data were recorded. In 75% of abortion cases in sheep and in 59% of cases in goats an etiologic diagnosis could be substantiated. Chlamydophila abortus is the most commonly involved agent in the etiology of caprine and ovine abortion (sheep 39%, goats 23%), followed by Toxoplasma gondii (sheep 19%, goats 15%) and Coxiella burnetti (sheep 1%, goats 10%). All other agents are of minor importance. An infectious cause of abortion based on histopathologic findings without isolation of agents was observed in sheep (10%) and goats (21%). Malformation occurred in sheep (2%) and goats (3%) and lesions suggestive for Vitamin E/Selenium deficiency were seen in goats only (2%).

Interruption of the cydB locus in Brucella abortus attenuates intracellular survival and virulence in the mouse model of infection.

Brucellosis is characterized by abortion in ruminants and a protracted undulant fever in humans, which often results in severe pathological manifestations. Scant information exists about the molecular mechanisms employed by Brucella abortus to combat host defenses or to persist and replicate within host cells. Transposon (Tn5) mutagenesis of B. abortus and the subsequent screening of mutants for sensitivity to killing in murine macrophages and in the mouse model led to the identification of mutants which were severely attenuated for intracellular survival. One group of mutants was interrupted in cydB, a gene that is part of the cydAB operon encoding cytochrome bd oxidase, which catalyzes an alternate terminal electron transport step in bacterial respiration. The elevated affinity for molecular oxygen of this enzyme in Escherichia coli has suggested that it is involved in the protection of sensitive enzymatic activities such as those of hydrogenases and nitrogenases from damage. B. abortus cydB::Tn5 strains exhibited heightened sensitivity to the respiratory inhibitors zinc and azide, highly reactive oxygen species such as hydrogen peroxide, low pH, and attenuated virulence in the mouse model of infection. Virulence was restored by an intact copy of cydAB or by B. abortus genes encoding the oxidative radical-scavenging enzyme Cu/Zn superoxide dismutase or catalase. These results suggest a bifunctional role for the products of the cydAB operon, both in preventing the buildup of oxidative free radicals and in detoxifying the intracellular compartment, thus indicating the importance of these products in preventing intracellular destruction. Intracellular conditions that favor expression of the cydAB operon are under investigation and may be linked to the acid sensitivity also observed in this strain.

Chlamydiose abortive des petits ruminants au Maroc : valeur épidémiologique d’un kit Elisar de <em>Chlamydophila abortus</em> (<em>Chlamydia psittaci</em> sérotype 1)

Le test Elisa recombinant (Elisar) utilisant la protéine de 80 à 90 kDa spécifique de Chlamydophila abortus, développé à l’Inra de Nouzilly en France, a été évalué au Maroc. Trois cent sept sérums appartenant à cinq groupes d’ovins et de caprins, provenant d’élevages aux taux d’avortements élevés ou bien présentés pour contrôle vétérinaire, ont été testés. La comparaison des résultats obtenus avec le test de fixation du complément (Tfc) a montré une grande discordance entre les deux techniques. En effet, seulement 16 sérums ont été positifs avec Elisar contre 130 positifs avec Tfc. En particulier, parmi les 55 sérums provenant d’animaux avortés, 48 ont été positifs avec Tfc et 16 avec Elisar. Ceci soulève avec acuité la problématique du rôle de Chlamydophila pecorum dans les avortements des petits ruminants au Maroc. La recherche différentielle des pathologies à Chlamydophila abortus et Chlamydophila pecorum est nécessaire pour préciser l’origine des divergences observées entre Elisar et Tfc.

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.

Preparation of a high titre Salmonella abortusovis H-antigen and antiserum and improvement of the microagglutination test

Serological diagnosis of small ruminant abortions due to Salmonella abortusovis (S.a.o) was improved by using modified procedures to produce high titre H-antigens and anti-H serum. While the titres of the two standard antigens were 1:8 and 1:20, both modified antigens had a titre of 1:50. Similarly, the use of adjuvanted and non adjuvanted antigens for the production of anti-H hyperimmune serum resulted in a titre of 1:160 000 as compared to 1:20 000 when standard procedures were used. In addition, the slow micro-agglutination test has been modified and results can now be read within the same day.

AFLP allows the identification of genomic markers of ruminant Chlamydia psittaci strains useful for typing and epidemiological studies

Amplified fragment length polymorphism (AFLP), a novel method for molecular typing, was evaluated for its ability to differentiate among a group of highly related Chlamydia psittaci strains isolated from ruminants and belonging to serotype 1. A total set of 12 strains were included in this study, 10 strains inducing abortion in ruminants and 2 strains from faecal samples. For the AFLP analysis, the total purified genomic DNA of each strain was submitted to a one-step digestion-ligation reaction for 3 h at 37 degrees C. DNA was digested with a single restriction endonuclease Mspl and ligated to specially constructed adapters. Subsequently, restricted fragments were selectively amplified under high stringency PCR conditions using primers complementary to the adapters. Amplified products were then resolved on agarose gel electrophoresis. The method is easy to perform, fast and reproducible. AFLP enabled characterization of C. psittaci strains at the infra-subspecific level. Thus, AFLP led to the identification of a cluster of strains on the basis of their AFLP patterns, constituted by French chlamydial isolates. It also permitted differentiation among strains in relation to host origin and to clinical syndromes. These data confirmed the highly discriminative power of AFLP towards the differentiation of closely related ruminant C. psittaci strains. The analysis will need to be applied to more samples to check the usefulness of AFLP markers in epidemiological and evolutionary studies.

Mouse Model for Central Nervous System Neospora caninum Infections

Neospora caninum is a protozoan parasite that causes severe disease in transplacentally infected dogs and abortions in domestic ruminants. Rodent models of neosporosis rely on treatment of hosts with methylprednisolone acetate (MPA) to enhance infections. The present study reports the development of an inbred BALB/c mouse model that results in central nervous system neosporosis in the absence of MPA treatment. Seven of 12 BALB/c mice died 26-70 days after subcutaneous (s.c.) inoculation with tachyzoites of the NC-1 strain of N. caninum, and none of 12 BALB/c mice died after s.c. inoculation with tachyzoites of the NC-3 strain. None of 8 HSD:ICR mice (4 mice, NC-1 strain; 4 mice, NC-3 strain) developed clinical neosporosis or died after s.c. inoculation with tachyzoites. Control BALB/c (2) and HSD:ICR (2) mice s.c. inoculated with Hanks' balanced salt solution did not develop clinical signs of disease. Some mice in all N. caninum-inoculated groups had brain lesions, but significantly (P < 0.05) more BALB/c mice inoculated with the NC-1 strain had brain lesions.

Protozoal Causes of Reproductive Failure in Domestic Ruminants

Protozoan parasites are a significant cause of abortion and infertility in domestic ruminants. Toxoplasma gondii, a widespread cause of abortion in sheep and goats, and Sarcocystis spp., which cause a common, frequently asymptomatic infection of domestic ruminants, both have a two-host life cycle. Carnivorous definitive hosts spread the infection through their feces and domestic ruminants are intermediate hosts. A similar, recently recognized protozoa, Neospora sp., has emerged as an important cause of reproductive disease, especially as an abortifacient in dairy cattle. Neospora is presumed to also have a two-host life cycle, although the definitive host(s) has not been identified. The venereally transmitted Tritrichomonas foetus is an important cause of pregnancy loss in naturally bred cattle throughout the world. In the absence of effective methods for vaccination or treatment, control of these parasites is based on management procedures to reduce infection and transmission.