Abortion In Small Ruminants Research Articles

Molecular Investigation of Small Ruminant Abortions Using a 10-Plex HRM-qPCR Technique: A Novel Approach in Routine Diagnostics.

The objective of this study was to apply and preliminarily evaluate a High-Resolution Melting (HRM) analysis technique coupled with qPCR, that allows the simultaneous detection of 10 different ruminant abortogenic pathogens, for investigating abortions in sheep and goats throughout Greece. A total of 264 ovine and caprine vaginal swabs were obtained the week following the abortion from aborted females and analyzed using a commercially available kit (ID Gene™ Ruminant Abortion Multiplex HRM, Innovative Diagnostics). Results indicated a high prevalence of Coxiella burnetii and Chlamydophila spp., which were detected in 48.9% and 42.4% of the vaginal swabs, respectively. Results for these most commonly detected pathogens were compared with those of a well-established commercial qPCR kit, with near-perfect agreement. Toxoplasma gondii, Salmonella spp., Brucella spp., Anaplasma phagocytophilum, Campylobacter fetus, and Neospora caninum were also identified, the two latter reported for the first time in the country in small ruminants. Mixed infections occurred in 35.6% of the animals examined. This technique allows for the simultaneous detection of many abortogenic pathogens in an accurate and cost-effective assay. Detection of uncommon or not previously reported pathogens in various cases indicates that their role in ovine and caprine abortions may be underestimated.

An 8000 years old genome reveals the Neolithic origin of the zoonosis Brucella melitensis

Brucella melitensis is a major livestock bacterial pathogen and zoonosis, causing disease and infection-related abortions in small ruminants and humans. A considerable burden to animal-based economies today, the presence of Brucella in Neolithic pastoral communities has been hypothesised but we lack direct genomic evidence thus far. We report a 3.45X B. melitensis genome preserved in an ~8000 year old sheep specimen from Menteşe Höyük, Northwest Türkiye, demonstrating that the pathogen had evolved and was circulating in Neolithic livestock. The genome is basal with respect to all known B. melitensis and allows the calibration of the B. melitensis speciation time from the primarily cattle-infecting B. abortus to approximately 9800 years Before Present (BP), coinciding with a period of consolidation and dispersal of livestock economies. We use the basal genome to timestamp evolutionary events in B. melitensis, including pseudogenization events linked to erythritol response, the supposed determinant of the pathogen’s placental tropism in goats and sheep. Our data suggest that the development of herd management and multi-species livestock economies in the 11th–9th millennium BP drove speciation and host adaptation of this zoonotic pathogen.

Molecular Detection of Coxiella burnetii in Vaginal Swab Samples from Sheep That Aborted.

Background: Coxiella burnetii, an obligate intracellular bacterium, is the etiological agent of Q fever in humans and one of the causes of abortion in small ruminants. Although coxiellosis is considered an exotic disease, there are a few reports in Mexico. Methods: The objective of this work was to determine the presence of C. burnetii DNA in vaginal samples from sheep that presented abortion and ram semen. A total of 180 vaginal exudate samples and 20 semen samples were obtained from five Central and Southern States of Mexico. Total DNA was extracted from vaginal swabs and C. burnetii was identified by PCR amplification and sequencing of the IS1111 insertion sequence. Results and Conclusion: In total, 110 (110/180) vaginal samples and 12 (12/20) semen samples were positive for C. burnetii. This is the first report of C. burnetii in sheep that aborted and in ram semen in Mexico.

Sheep abortions associated with Neospora caninum and Toxoplasma gondii infections in multiple flocks from Southern Brazil.

Neosporosis and toxoplasmosis are important parasitic causes of abortions in small ruminants. This study verified the occurrence of these diseases in sheep fetuses from Santa Catarina State, Southern Brazil from 2015 to 2022. Sheep fetuses were necropsied with organ sampling for histopathology, and polymerase chain reaction (PCR) for Neospora caninum and Toxoplasma gondii using the Nc5 and SAG2 targets, respectively, in frozen brain tissue. Microbiological culture and RT-PCR for Pestivirus were conducted to discard other abortion causes. One positive fetus for toxoplasmosis was genotyped using multiplex multilocus nested PCR-RFLP (Mn-PCR-RFLP) with ten genetic markers. Fifty-five sheep fetuses were evaluated, with 10 (18.2%) cases of neosporosis and 7 (12.7%) cases of toxoplasmosis, comprising six and four flocks, respectively. Macroscopically, neosporosis abortions exhibited fetal mummification, maceration, and arthrogryposis. Toxoplasmosis abortions showed fetal mummification and maceration. The neosporosis abortions included lymphoplasmacytic myositis (70%; 7/10) and myocarditis (60%; 6/10), in addition to necrotizing encephalitis and gliosis (50%; 5/10). Toxoplasmosis abortions included lymphoplasmacytic necrotizing encephalitis (71.4%; 5/7), lymphoplasmacytic myositis (42.8%, 3/7), and myocarditis (14.3%; 1/7). Through PCR, N. caninum and T. gondii were detected in 6 (60%) and 5 (71.4%) fetuses, respectively. In one fetus, T. gondii genotyping was conducted, which was characterized as atypical genotype ToxoDB #98. All of the cases were negative for Pestivirus and bacterial agents. This study establishes the occurrence of these diseases as causes of abortions, malformations, mummification, and fetal maceration in sheep, with the characterization of an atypical T. gondii genotype in one of the fetuses.

Seroepidemiology of infectious agents associated with abortion in small ruminants

Seroepidemiology of infectious agents associated with abortion in small ruminants

A Retrospective Investigation of Abortion Storm in Abergele Goats, Waghimira Zone, Amhara Region, Ethiopia.

A retrospective study was conducted in Abergele and Ziquala districts in Ethiopia to investigate the occurrence, impact, and potential risk factors for abortion in small ruminants linked to a regional abortion storm. Affected (case) and nonaffected (control) villages were compared to assess infectious and noninfectious risk factors causing abortions. A case village was defined as a village with abortion seen in all households enrolled in the study, while a control village is characterized by presence of abortion in two and fewer households. A questionnaire survey, focus group discussions, and serological examinations were used to assess the differences in the abortion rate between the case and control villages. The Rose Bengal Plate Test for Brucellosis, Toxoplasma gondii Antibody Test Kit for Toxoplasma gondii, and ELISA for Chlamydophila abortus and Coxiella burnetii were used to detect antibodies. Per village 15 household flocks were selected. In the case villages, all flocks were affected by abortion (a mean abortion of 13 animals in Abergele and 9 in Ziquala). In contrast, only three households reported abortions in the control villages. A total of 176 blood samples were collected from the case and control villages for further laboratory diagnosis of possible causes of abortion. Of the examined flocks, 17%, 10.2%, and 2.8% were positive for Coxiella burnetii, Toxoplasma gondii, and Chlamydophila abortus, respectively. However, antibodies against Brucella melitensis were not detected. While the seroprevalence was greater for some infectious agents of abortion, there was no significant difference between the case and control villages. It is clear that the abortion problem in the study villages is complex and likely due to a mix of poor husbandry practices and the presence of infectious diseases. To better understand the underlying causes of abortion, there is a need to conduct a longitudinal study involving testing for more pathogens at the household level combined with reliable data on husbandry practices.

Molecular investigation of Coxiella burnetii in aborted fetus of small ruminants in southeast Iran.

The domestic animal, known as a main reservoir of Coxiella burnetii, is susceptible to the occurrence of coxiellosis, which can lead to abortions in domestic animals, causing significant economic damage and posing risks to human health. Therefore, the purpose of this study is to investigate C. burnetii as the causative agent of Q fever in abortion samples of small ruminants in southeastern Iran. This study was conducted between 2020 and 2021 in Zarand city, located in Kerman province (southeast Iran). In this study, 50 abomasum swab samples of aborted sheep and goat fetuses were collected and analyzed using molecular methods to identify C. burnetii. The results revealed that 26% (n: 13) of the collected abortion samples were infected with C. burnetii. Among the positive samples, two (50%) belonged to goat abortion samples while 11 (23.9%) belonged to sheep abortion samples. This study demonstrates that C. burnetii is one of the causes of abortion in small ruminants in southeastern Iran. It is recommended to pay more attention to C. burnetii in domestic animals due to its significant economic impact on livestock and its potential implication for human health in Iran.

Seroprevalence and assessment of public awareness of Brucella spp., Toxoplasma gondii and Chlamydia abortus in small ruminants from selected smallholder commercial farms of Zimbabwe.

Brucella spp., Toxoplasma gondii, and Chlamydia abortus have long been recognized as zoonoses and significant causes of reproductive failure in small ruminants globally. A cross-sectional study was conducted in August 2020 to determine the seroprevalences of Brucella spp., Toxoplasma gondii and Chlamydia abortus in 398 small ruminants from four districts of Zimbabwe (Chivi, Makoni, Zvimba, and Goromonzi) using Indirect-ELISAs. A structured questionnaire was used to assess the knowledge, attitudes, and practices of 103 smallholder farmers towards small ruminant abortions, Brucella spp., T. gondii and C. abortus, and to obtain a general overview of the significance of small ruminant reproductive failure(s) on their livelihoods. The overall seroprevalences were: 9.1% (95% CI: 6.4-12.3) for Brucella spp., 6.8% (95% CI: 4.5-9.7) for T. gondii and 2.0% (95% CI: 0.9-3.9) for C. abortus. Location, age, parity, and abortion history were associated with Brucella spp. seropositivity. Location was also associated with both T. gondii and C. abortus seropositivity. The questionnaire survey established that 44% of respondents had recently faced reproductive disease challenges within their flocks, with 34% correctly identifying abortion causes and only 10%, 6% and 4% having specific knowledge of Brucella spp., C. abortus and T. gondii, respectively. This study provides the first serological evidence of Brucella spp. in small ruminants since 1996 and builds the evidence on small ruminant toxoplasmosis and chlamydiosis in Zimbabwe. Evidence of these zoonoses in small ruminants and the paucity of knowledge shows the need for a coordinated One Health approach to increase public awareness of these diseases, and to establish effective surveillance and control measures. Further studies are required to establish the role these diseases play in small ruminant reproductive failure(s), to identify the Brucella spp. detected here to species/subspecies level, and to assess the socio-economic impact of reproductive failure in livestock among marginalised rural communities.

O-072 Infectious agents of abortion in small ruminants between 2012 and 2022

O-072 Infectious agents of abortion in small ruminants between 2012 and 2022

Histopathological analysis of placental lesions caused by Chlamydia abortus 1B vaccine strain in vaccinated ewes

Background Chlamydia abortus is one of the most diagnosed causes of infectious abortion in small ruminants. Infections can be controlled using the live, attenuated C. abortus strain 1B vaccine, which has been associated with infection and abortion in animals. This study aimed to compare the severity and the distribution of lesions caused by this vaccine strain (vt) with those resulting from a wild-type (wt) infection. Methods Two grossly affected and 1B-positive (by qPCR and RFLP analysis) placentas from a vaccinated sheep flock were analysed. Histopathological lesions and immunohistochemical labelling (IHC) were graded (increasing score from 0 to 5) according to their severity and distribution. Pathology in the vt infected placentas was compared with that in two wt infected placentas. Datasets generated for observed histological and pathological features were analysed using principal component analysis (PCA). Results Histopathologically, the lesions in both vt and wt-placentas presented as typical multifocal necrosuppurative placentitis, associated with vasculitis, mural necrosis, and thrombosis. IHC for C. abortus revealed intense staining with a multifocal distribution in most cotyledons in both vt and wt placentas. Comparison of the pathological lesions between vt and wt by PCA revealed a similar distribution and severity, revealing a strong association with features such as necrosis and inflammatory infiltration between vt and wt placentas. A weaker association with IHC was observed. Conclusion This study shows that the C. abortus attenuated 1B vaccine strain presents no significant differences in severity and distribution of pathological lesions from those typically observed following a wt infection.

Toxoplasmosis in Zoo Animals: A Retrospective Pathology Review of 126 Cases.

Simple SummaryToxoplasma gondii is widespread amongst domestic animals and can affect humans. Whilst the disease is of economic importance and well-studied in ruminants, this is the first large-scale study evaluating toxoplasmosis in zoo animals. The aim of this study was to identify species that are particularly susceptible to disease, review clinical symptoms, organ distribution and key target tissues, highlight unusual outbreaks/presentations, assess predilections, evaluate toxoplasmosis as the cause of death and consider seasonality. A total of 31 species were represented, with ring-tailed lemurs (RTLs), meerkats, Pallas’ cats and squirrel monkeys most affected. An unusual outbreak occurred in Asian short-clawed otters. Non-specific, neurological, gastrointestinal, respiratory signs and sudden death dominated clinically, mainly in animals over 12 months of age that presented with systemic disease. Pallas’ cats showed encephalitis (inflammation of the brain) and lymphoid tissue was frequently involved in RTLs. Lesions were primarily found in heart, liver, lungs, brain, spleen and lymph nodes. Cases occurred year-round, with species-specific peaks and increases between August and November. This study demonstrates that toxoplasmosis is a significant cause of disease in zoo animals. Knowledge of species susceptibility will aid the treatment of affected animals by zoo veterinarians. Due to the lifecycle of the parasite, feral cat/rodent control is crucial to reduce infection pressure and prevent disease.Toxoplasma gondii is an extremely successful zoonotic protozoan parasite that has been demonstrated in a wide range of endo- and poikilothermic species. Although infection is widespread amongst domestic animals, overt disease other than abortion in small ruminants is sporadic. This survey evaluates toxoplasmosis in zoo animals based on a systematic review of pathology archive material (n = 33,506 submissions) over a 16-year study period. A total of 126 submissions, deriving from 32 zoos, two educational facilities and two private owners, were included in the study, based on gross lesions, cytological, histological and immunohistological diagnosis of toxoplasmosis. Clinical history, signalment, annual distribution and post-mortem findings were evaluated. A total of 31 species (mammalian 97%/avian 3%) were represented in the study material. Ring-tailed lemurs, slender tailed meerkats, Pallas’ cats, and squirrel monkeys were most affected. An unusual outbreak occurred in Asian small-clawed otters, in which toxoplasmosis has not been reported to date. Clinically, animals over 12 months of age presented with non-specific symptoms (anorexia, weight loss, lethargy, debilitation), neurological, gastrointestinal or respiratory signs and sudden death. Systemic disease predominated, with a propensity for encephalitis in meerkats and Pallas’ cats and systemic disease involving lymphoid tissues in ring-tailed lemurs. Cases in the UK occurred year-round, with species-specific peaks and increases between August and November. This study reinforces the importance of toxoplasmosis as a significant cause of sporadic and epizootic mortalities in a wide range of zoo animals. Feral cat control is crucial to reduce infection pressure.

Investigation of Toxoplasma gondii and Neospora caninum as cause of ovine abortion in affected flocks of Urmia, northwest of Iran

Toxoplasma gondii and Neospora caninum, two obligatory intracellular protozoan parasites, are important causes of neonatal mortality and abortion in farmed ruminants worldwide. Previously, protozoan abortions in small ruminants were attributed to T. gondii, but the role of N. caninum in these abortions was uncertain. The aim of this study was to investigate the role of T. gondii and N. caninum in ovine abortion in Urmia, northwest of Iran using a molecular method. Overall, 130 placenta and brain samples of aborted ovine foetuses were collected. Extracted DNA from placenta and CNS tissues of the aborted foetuses were analysed using PCR with primers specific for T. gondii and N. cani­num. The association of the frequency of T. gondii and N. caninum infection of aborted foetuses with age and breed in flocks was also studied. The results showed that out of the 130 examined ovine foetuses, 5.3 and 2.3 % were PCR-positive for T. gondii and N. caninum DNA, respectively. In this study, no significant differences were recorded relating to Toxoplasma and Neospora infection in different age groups in flocks and among sheep breeds included in the present study (P>0.05). The results of this study proved the importance of T. gondii and N. caninum as reasons of abortion in the studied area.

Molecular investigation of most important viruses causing abortion in small ruminants: the importance of akabane virus

Objective. This study was aimed to investigate the presence of pestivirus, schmallenberg virus (SBV), akabane virus (AKAV), and determined the seasonal distribution in small ruminant abortion cases Materials and Methods. A total of 164 small ruminant aborted fetuses (39 goats and 125 sheep) were investigated in West Turkey between the years of 2015 and 2019. While the SBV and pestivirus were examined with real-time RT-PCR, the AKAV was examined with nested-PCR. Results. In this study, while four sheep (2.3%) were found to be pestivirus positive, nine sheep and one goat (5.9%) were found to be AKAV positive. SBV was detected in none of the animals. According to the results of the studies carried out, AKAV, a vector-borne virus, was detected more than other viruses in sheep and goat abortions in West Turkey. A positive akabane virus sample detected in sheep fetuses was sequenced based on the partial S segment. This sample showed 99-100% similarity to the Adana-15, Aksu-1, and Aksu-2 isolates previously detected in Southern Turkey. Phylogenetic analysis showed that the sequenced isolate in this study was clustered within genogroup Ib. Another important piece of data revealed in this study was the determination of the seasonal distribution of sheep and goat abortions. Abortion cases which started to increase in November reached a peak in February and declined till August. Conclusions. This is the first study in the Aegean Region (including seven provinces) in which three important viral agents were simultaneously investigated in small ruminant aborted fetuses.

An Accessible Diagnostic Toolbox to Detect Bacterial Causes of Ovine and Caprine Abortion.

Results of laboratory investigations of ovine and caprine cases of abortion in the lambing season 2015–2016 were analyzed, using pathology records of submissions to Royal GD (Deventer, the Netherlands) from January until and including April 2016, in comparison with the results of two accessible alternative techniques for sampling aborted lambs and kids, swabbing the fetal oropharynx and puncture of the fetal lung. Chlamydia abortus was the main cause of abortion in sheep as well as in goats. Other causes of abortion were Campylobacter spp., Listeria spp., Escherichia coli, and Yersinia enterocolitica. Ovine pathological submissions resulted more often in detecting an infectious agent compared to caprine submissions. For the three main bacterial causes of abortion, Campylobacter spp., Listeria spp., and Chlamydia spp., compared to results of the pathological examination, oropharynx mucus, and fetal lung puncture samples showed an observed agreement of 0.87 and 0.89, an expected agreement of 0.579 and 0.584, and a kappa value of 0.691 and 0.737 (95% CI: 0.561–0.82 and 0.614–0.859), respectively. The agreement between the results of the pathological examination and both fetal lung puncture and oropharynx mucus samples was classified as good. In conclusion, although a full step-wise post-mortem examination remains the most proper way of investigating small ruminant abortions, the easily accessible, low-threshold tools for practitioners and farmers as described in this paper not only provide reliable results compared to results of the post-mortem examination but also stimulates farmers and veterinarians to submit fetuses and placentas if necessary. Suggestions for further improvement of both alternatives have been summarized. Both alternatives could also be tailor-made for specific regions with their specific causes of abortion.

Seroprevalence of brucellosis and chlamydiosis in sheep and goats with history of abortion in Iran

• Brucellosis and chlamydiosis are common causes of abortion in livestock. • The prevalence of brucellosis was significantly higher in goats compared to sheep. • Inversely, goats showed a lower prevalence of chlamydiosis compared to sheep. • Brucella melitensis biovars 1 was identified in sheep and goats aborted fetuses. Abortion in small ruminants is a critical issue in many developing countries including Iran and causes devastating economic losses in small ruminant industry. The aim of this study was to evaluate the seroprevalence of brucellosis and chlamydiosis in sheep and goats with a history of abortion during lambing seasons in Iran. Samples were collected during 2019 from sheep and goat flocks located in Fars province (Southern Iran). A total of 124 sera (101 sheep and 23 goats) from aborted females as well as 29 aborted fetuses were sampled and analyzed for Chlamydia abortus and Brucella spp. using indirect enzyme-linked immunosorbent assay (i-ELISA) for chlamydiosis and a set of serological (RBT, SAT and 2-ME and i-ELISA) and bacterial tests for brucellosis. Seropositivity to C. abortus was also detected in 21.7 % and 4.3 % of sheep and goats, respectively. Brucella specific antibodies were found in 29.1 %, 22.6 %, 20.9 % and 23.4 % of tested sheep and goats using RBT, SAT,2-ME and i-ELISA, respectively. Bacterial isolation and biotyping led to the characterization of B. melitensis biovars 1 in sheep and goats aborted fetuses of Fars province. High prevalence of infection associated with brucellosis and chlamydiosis in both sheep and goat with a history of abortion highlights risks of human zoonotic infections by these pathogens. Therefore, special attention is required for further study and control program to counteract these infections in animals and humans.

Eleven years of Toxoplasma gondii serological follow-up in a goat herd and association of toxoplasmosis with reproductive losses.

Toxoplasmosis is considered one of the most important causes of abortion in small ruminants. The aim of this study was to evaluate the relationship between Toxoplasma gondii antibody titres and reproductive losses over an 11year period in a goat farm located in Buenos Aires province, Argentina. Blood samples were obtained from 85 goats, representing three breeds, during the last third of gestation (n=165 gestations), in consecutive pregnancies (2008-2019), and from 51 goats during kidding to analyze seroconversion. Serum was evaluated by IFAT with T. gondii antigen, using 1:100 dilution as the cut-off titre and processed to end titre. An overall reproductive loss of 31% (51/165) was detected, including 16.4% (27/165) abortions and 14.6% (24/165) perinatal deaths. The seropositivity to T. gondii was 100% (85/85) with all animals positive in successive samplings and, therefore, considered chronically infected. Antibody titres showed average values greater than 1100 in each year and breed group. Differences in antibody levels were associated with breed and were lower in those that were predominately Creole and higher in those that were predominately Saanen. Seroconversion was detected in 16.2% (6/37) and 57.1% (8/14) of goats from the Creole and Sannen breed groups, respectively. There were no significant differences in the antibody titre average between goats with reproductive losses and those with healthy kids, although the goats with perinatal deaths had a significantly higher titre average. These results suggest reinfection or reactivation, although no association with reproductive losses was observed. Higher antibody titres were associated with perinatal deaths. The high T. gondii antibody titres in a farm with 100% seroprevalence did not allow for association with reproductive losses, particularly abortion, to be assessed.

Infectious Abortions in Small Ruminants: Challenges for Diagnosis and Public Health.

In this study, we report an abortion outbreak in a ruminant herd consisting of goats, sheep, and cows, with scenarios in two consecutive years. In early 2017, abortions occurred in ∼70% of goats and 66% tested positive for Coxiella burnetii (C. burnetii) and 40% of goats were positive for Chlamydophila abortus (C. abortus). In February 2018, the same herd reported an abortion rate of 75%, with 55% positive for C. burnetii, 36% for C. abortus, and 22% for Toxoplasma gondii. Six goat milk samples were positive for C. burnetii by molecular analysis. Three family members were positive for C. burnetii. C. burnetii could be considered as the main cause of abortions in the first and second year. Animals that undergo an infection and abortion are prone to secondary infections. Vaccination or other rapid interventions should be initiated to protect animals and humans.

Identification of Main Brucella species Implicated in Ovine and Caprine Abortion Cases by Molecular and Classical Methods.

Brucellosis is recognized as a major public health concern leading to critical economic losses in livestock animals. The present study assessed Brucella spp. isolated from aborted ovine and caprine fetuses in different parts of Iran between 2016 and 2019. It used classic and molecular methods in order to determine the Brucella species carrying higher risks of abortion complications in these animals. A total of 189 samples from 35 cases/case series from milk (16 sheep, and 8 goats), 19 abomasum content (sheep), and 146 aborted fetuses (116 sheep, and 30 goats) were bacteriologically examined. Subsequently, the resultant Brucella isolates were further characterized by phenotypic and molecular approaches. The multiplex Polymerase chain reaction (PCR) (Bruce-ladder) and IS711-based PCR were performed on all the extracted DNA to evaluate the presence of Brucella spp. As suggested by the obtained results, all recovered isolates from ovine and caprine abortion samples were either B. melitensis or B. abortus. An issue of concern was the implication of B. melitensis vaccine strain Rev1 in a small portion of sheep and goat abortion cases. Despite the recent B. abortus burden in ovine, aborted cases were predominantly associated with B. melitensis infections in both ovine and caprine, and B. melitensis biovar 1 was responsible for the majority of studied cases. These data and the techniques implemented in the present study can shed light on the level of implication of different Brucella species in ovine and caprine abortion in Iran. The present study identified Brucella agents responsible for abortion in small ruminants at the biovar level. Therefore, it provides precious information for future control programs and vaccination strategies in Middle Eastern regions.

Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks

BackgroundToxoplasma gondii is a major cause of abortion in small ruminants and presents a zoonotic risk when undercooked meat containing cysts is consumed. The aim of the present study was to investigate the genetic diversity among the T. gondii strains circulating in ovine livestock in Spain.MethodsSelected samples collected from abortion outbreaks due to toxoplasmosis (n = 31) and from chronically infected adult sheep at slaughterhouses (n = 50) in different Spanish regions were bioassayed in mice, aiming at parasite isolation. In addition, all original clinical samples and the resulting isolates were genotyped by multi-nested PCR-RFLP analysis of 11 molecular markers and by PCR-DNA sequencing of portions of the SAG3, GRA6 and GRA7 genes.ResultsAs a result, 30 isolates were obtained from 9 Spanish regions: 10 isolates from abortion-derived samples and 20 isolates from adult myocardial tissues. Overall, 3 genotypes were found: ToxoDB#3 (type II PRU variant) in 90% (27/30) of isolates, ToxoDB#2 (clonal type III) in 6.7% (2/30), and ToxoDB#1 (clonal type II) in 3.3% (1/30). When T. gondii-positive tissue samples (n = 151) were directly subjected to RFLP genotyping, complete restriction profiles were obtained for 33% of samples, and up to 98% of the specimens belonged to the type II PRU variant. A foetal brain showed a clonal type II pattern, and four specimens showed unexpected type I alleles at the SAG3 marker, including two foetal brains that showed I + II alleles as co-infection events. Amplicons of SAG3, GRA6 and GRA7 obtained from isolates and clinical samples were subjected to sequencing, allowing us to confirm RFLP results and to detect different single-nucleotide polymorphisms.ConclusionsThe present study informed the existence of a predominant type II PRU variant genotype (ToxoDB#3) infecting domestic sheep in Spain, in both abortion cases and chronic infections in adults, coexisting with other clonal (ToxoDB#1 and ToxoDB#2), much less frequent genotypes, as well as polymorphic strains as revealed by clinical sample genotyping. The use of multilocus sequence typing aided in accurately estimating T. gondii intragenotype diversity.

Evidence that Chlamydia abortus vaccine strain 1B produces indistinguishable placental lesions to wild type strains

Chlamydia abortus is the most commonly diagnosed cause of abortion in small ruminants around the world [1]. Control of chlamydial abortion is achieved in several European countries using an attenuated live 1B C. abortus vaccine strain, which can be distinguished from virulent wild-type strains by PCR-RFLP analysis [2]. Application of this method has provided molecular evidence that the 1B strain can cause abortion in ewes [3, 4]. The objective of this study was to define the distribution of lesions and bacterial load in cotyledons from ewes vaccinated with the 1B strain compared to normal wild-type infections. A Chlamydia-free flock of 75 multiparous adult ewes were vaccinated twice, two years apart, each prior to mating, with the commercial 1B vaccine. In the second lambing season following the last vaccination, placentae (n=116) were collected and analysed by C. abortus real-time qPCR [3]. Only two of the placentae, both from the same ewe, were found to be positive. Viable organisms were isolated from these placentae and confirmed by RFLP-PCR [3] to be vaccine-type. All cotyledons from these placentae were analysed by histopathology and immunohistochemistry [5], and compared with those from wild-type infected placentae. The lesions in the vaccine-type infected placentae were indistinguishable from the wild-type infected placentae in terms of their severity, load and distribution. These results suggest that the 1B vaccine strain of C. abortus can cause chlamydial abortion in ewes producing typical placental lesions to wild-type infected animals, and could be circulating with the potential to cause natural infection and disease.