Transmission of Actinobacillus pleuropneumoniae among weaned piglets on endemically infected farms

Clinical outbreaks due to Actinobacillus pleuropneumoniae occur recurrently, despite the wide-scale use of antimicrobials or vaccination. Therefore, new approaches for the prevention and control of these outbreaks are necessary. For the development of alternative measures, more insight into the transmission of the bacterium on farms is necessary. The aim of this cohort study was to quantify transmission of A. pleuropneumoniae amongst weaned piglets on farms. We investigated three possible transmission routes: (i) indirect transmission by infected piglets within the same compartment, (ii) transmission by infected pigs in adjacent pens and (iii) transmission by direct contact within pens. Additionally, we evaluated the effect of independent litter characteristics on the probability of infection. Two farms participated in our study. Serum and tonsil brush samples were collected from sows pre-farrowing. Serum was analysed for antibodies against Apx toxins and Omp. Subsequently, tonsil brush samples were collected from all piglets from these dams (N = 542) in three cohorts, 3 days before weaning and 6 weeks later. Tonsil samples were analysed by qPCR for the presence of the apxIVA gene of A. pleuropneumoniae. Before weaning, 25% of the piglets tested positive; 6 weeks later 47% tested positive. Regression and stochastic transmission models were used to assess the contribution of each of the three transmission routes and to estimate transmission rates. Transmission between piglets in adjacent pens did not differ significantly from that between non-adjacent pens. The transmission rate across pens was estimated to be 0.0058 day⁻¹ (95% CI: 0.0030–0.010), whereas the transmission rate within pens was ten times higher 0.059 day⁻¹ (95% CI: 0.048–0.072). Subsequently, the effects of parity and serological response of the dam and litter age at weaning on the probability of infection of pigs were evaluated by including these into the regression model. A higher dam ApxII antibody level was associated with a lower probability of infection of the pig after weaning; age at weaning was associated with a higher probability of infection of the pig after weaning. Finally, transmission rate estimates were used in a scenario study in which the litters within a compartment were mixed across pens at weaning instead of raising litter mates together in a pen. The results showed that the proportion of infected piglets increased to 69% if litters were mixed at weaning, indicating that farm management measures may affect spread of A. pleuropneumoniae.     

Enhancement of protective immune responses by oral vaccination with Saccharomyces cerevisiae expressing recombinant Actinobacillus pleuropneumoniae ApxIA or ApxIIA in mice

We previously induced protective immune response by oral immunization with yeast expressing the ApxIIA antigen. The ApxI antigen is also an important factor in the protection against Actinobacillus pleuropneumoniae serotype 5 infection; therefore, the protective immunity in mice following oral immunization with Saccharomyces cerevisiae expressing either ApxIA (group C) or ApxIIA (group D) alone or both (group E) was compared with that in two control groups (group A and B). The immunogenicity of the rApxIA antigen derived from the yeast was confirmed by a high survival rate and an ApxIA-specific IgG antibody response (p < 0.01). The highest systemic (IgG) and local (IgA) humoral immune responses to ApxIA and ApxIIA were detected in group E after the third immunization (p < 0.05). The levels of IL-1β and IL-6 after challenge with an A. pleuropneumoniae field isolate did not change significantly in the vaccinated groups. The level of TNF-α increased in a time-dependent manner in group E but was not significantly different after the challenge. After the challenge, the mice in group E had a significantly lower infectious burden and a higher level of protection than the mice in the other groups (p < 0.05). The survival rate in each group was closely correlated to the immune response and histopathological observations in the lung following the challenge. These results suggested that immunity to the ApxIA antigen is required for optimal protection.     

Humoral immune responses to Mycoplasma hyopneumoniae in sows and offspring following an outbreak of mycoplasmosis

Previously healthy sows, seropositive to Mycoplasma hyopneumoniae, developed clinical signs of mycoplasmosis, as well as increasing amounts of antibodies to M. hyopneumoniae during an outbreak of the disease in a herd. During the early phase of the outbreak, young piglets (2 weeks) with maternal antibodies remained healthy while older seronegative piglets (4–7 weeks) developed the disease. The duration of the maternal antibodies to M. hyopneumoniae varied between litters and was related to the amount of antibodies in the serum of the dam. In sows, the level of serum antibodies decreased continuously from 4 weeks ante partum to partus, and the level of antibodies in the whey of colostrum was comparable to that in serum 4 weeks ante partum. After loss of maternal antibodies to M. hyopneumoniae, seropositive animals were not found among piglets younger than 9 weeks. Therefore peripheral blood mononuclear cells (PBMC) were collected from various age categories of piglets in order to measure the ability to produce antibodies to M. hyopneumoniae in vitro. PBMC obtained from piglets aged 1 and 3 weeks produced few antibodies to M. hyopneumoniae. Significantly higher levels of antibodies to M. hyopneumoniae were produced by PBMC obtained from pigs aged 5–9 weeks. Thus, the ability of PBMC to produce antibodies to M. hyopneumoniae in vitro seemed to be age-dependent.     

Pleuritis in slaughter pigs: Relations between lung lesions and bacteriology in 10 herds with high pleuritis

Pleuritis in slaughter pigs has increased in recent years in the Netherlands. The aim of the present study was to determine what respiratory pathogens were involved in pleuritis.In total, lungs of 968 slaughter pigs from 10 herds with high prevalence of pleuritis were morphologically examined for size, location, and type of lesions. Moreover, histology and bacteriology were performed.Examination of gross lung lesions showed 45% pleuritis, 14% pleuropneumonia and 38% catarrhal pneumonia. Peribronchiolar cuffing was found in 61 of 142 samples. Actinobacillus pleuropneumoniae was cultured from 22 lung samples from four herds. Pasteurella multocida was cultured from 55 lung samples in eight herds. No specific pattern with respect to the causal pathogens was found.In conclusion, no single infectious cause of pleuritis was found. A variety of infectious agents combined with environmental factors should be considered as a cause of pleuritis.     

Molecular heterogeneity of plpE gene in Indian isolates of Pasteurella multocida and expression of recombinant PlpE in vaccine strain of P. multocida serotype B: 2

Outer membrane proteins of Pasteurella (P.) multocida have been known to be protective immunogens. Pasteurella lipoprotein E (PlpE) has been reported to be an important cross reactive outer membrane protein in P. multocida. The gene encoding the PlpE of P. multocida serotypes A: 3, B: 2 and D: 1 was amplified from the genomic DNA. The amplified products were cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clones revealed a single open reading frame of 1,011 bp, 1,008 bp and 1,017 bp encoding a protein with a calculated molecular mass of 37.829 kDa, 37.389 kDa and 37.965 kDa for serotypes A: 3, B: 2 and D: 1 respectively. The comparison of the plpE sequence in different capsular types revealed a high degree (>90%) of homology. Furthermore, the plpE gene of Haemorhhagic septicaemia causing serotype (B: 2) was expressed in E. coli and recombinant PlpE was strongly immunostained by antiserum against whole cell antigen, indicating that the protein is expressed in vivo.     

CpG oligodeoxynucleotides augment the immune responses of piglets to swine Pasteurella multocida living vaccine in vivo

Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) prevent development of T-helper type 2 (Th2) immune response and reverse established allergic responses in mouse models. However, little work on immune responses in piglets has been conducted in vivo. In this report, the ability of a porcine-specific CpG ODN to act as an immunostimulant and enhance immune responses of piglets to swine Pasteurella multocida living vaccine (SPML vaccine) was determined. The titre of IgG and IgG1/IgG2 isotype to SPML vaccine in serum, the proliferation of lymphocytes, SPML-specific interferon-gamma (IFN-gamma) and IL-6, TNF-alpha, IL-4 production of PBMCs in vitro and IFN-gamma, IL-6, TNF-alpha, IL-4, IL-10 in piglets serum were examined to identify the immune responses of the piglets. Immune responses of the piglets vaccinated with SPML and CpG ODN were significantly stronger than responses of piglets vaccinated with SPML alone. All these data summarized that immunostimulatory CpG ODN could modulate the immune response towards a Th1-like response when co-administered to piglets during SPML vaccination, which suggested that the therapeutic uses envisioned for these ODNs (as vaccine adjuvants and immunoprotective agents) may be applicable to husbandry animals.     

Effects of salinomycin and Bacillus subtilis on growth performance and immune responses in broiler chickens

The present study was undertaken to compare the effect of salinomycin and Bacillus subtilis on growth performance, serum antibody levels against Clostridium spp. and Eimeria spp., and cytokine mRNA expression levels in broiler chickens raised in the used litter. Broiler chickens fed a diet containing salinomycin showed lower (P < 0.05) body weights compared with the control diet-fed counterparts. Serum nitric oxide levels were significantly (P < 0.05) elevated in chickens fed the B. subtilis-enriched diet compared with those on either the salinomycin-fed or control diet-fed chickens. None of the dietary treatments affected (P > 0.05) serum antibody levels against Clostridium perfringens toxins. Both salinomycin and B. subtilis significantly lowered (P < 0.05) the serum levels of Eimeria-specific antibodies compared with the control group. Salinomycin, but not B. subtilis, significantly modulated (P < 0.05) the expression of cytokines encoding interferon-γ (IFN-γ), interleukin10 (IL-10) and tumor necrosis factor superfamily 15 (TNFSF15) compared with the control group. In conclusion, dietary salinomycin and B. subtilis affected serum anticoccidial antibody and intestinal cytokine expression, but failed to improve growth performance in broiler chickens. Further study is warranted to investigate the mode of action of salinomycin on host immune response and growth performance in broiler chickens.     

Estimation of sensitivity, specificity and predictive values of two serologic tests for the detection of antibodies against Actinobacillus pleuropneumoniae serotype 2 in the absence of a reference test (gold standard)

Latent-class models were used to determine the sensitivity, specificity and predictive values of a polyclonal blocking enzyme-linked immunosorbent assay (ELISA) and a modified complement-fixation test (CFT) when there was no reference test. The tests were used for detection of antibodies against Actinobacillus pleuropneumoniae serotype 2 in a survey of respiratory diseases in Danish finishing pigs. The estimates were obtained by maximum-likelihood and also by a Bayesian method (implemented with Gibbs sampling). Possible dependence of diagnostic errors was investigated by comparing models where independence was assumed to models allowing for conditional dependence, given the true disease status.No strong evidence of conditional dependence in either test sensitivity or specificity was found. Assuming independence, maximum-likelihood estimates and 95% confidence intervals of the sensitivity and specificity of the ELISA were 100% and 92.8% (90.1–95.5%) and the corresponding values of the CFT were 90.6% (85.8–95.4%) and 98.6% (98.0–99.3%), respectively. Bayesian estimates and posterior 95% credible intervals of the sensitivity and specificity of the ELISA were 99.7% (98.7–100%) and 92.7% (89.9–95.3%) and of the CFT were 90.6% (86.0–95.3%) and 98.7% (98.0–99.3%). The sensitivity and specificity of a combined test, where the CFT is subsequently applied to the pig sera that test positive in the ELISA, were estimated at 90.2% (85.6–95.0%) and 99.9% (99.8–100%), respectively. The cost of the combined test was less than the cost of the use of the CFT alone, at prevalences <54%. Prevalences and predictive values and their 95% limits were estimated in six sub-samples of data. The estimates of sensitivity and specificity obtained in the present investigation generally validate those reported from other sources.     

Elucidating the role of ApxI in hemolysis and cellular damage by using a novel apxIA mutant of Actinobacillus pleuropneumoniae serotype 10

Exotoxins produced by Actinobacillus (A.) pleuropneumoniae (Apx) play major roles in the pathogenesis of pleuropneumonia in swine. This study investigated the role of ApxI in hemolysis and cellular damage using a novel apxIA mutant, ApxIA336, which was developed from the parental strain A. pleuropneumoniae serotype 10 that produces only ApxI in vitro. The genotype of ApxIA336 was confirmed by PCR, Southern blotting, and gene sequencing. Exotoxin preparation derived from ApxIA336 was analyzed for its bioactivity towards porcine erythrocytes and alveolar macrophages. Analysis results indicated that ApxIA336 contained a kanamycin-resistant cassette inserted immediately after 1005 bp of the apxIA gene. Phenotype analysis of ApxIA336 revealed no difference in the growth rate as compared to the parental strain. Meanwhile, ApxI production was abolished in the bacterial culture supernatant, i.e. exotoxin preparation. The inability of ApxIA336 to produce ApxI corresponded to the loss of hemolytic and cytotoxic bioactivity in exotoxin preparation, as demonstrated by hemolysis, lactate dehydrogenase release, mitochondrial activity, and apoptosis assays. Additionally, the virulence of ApxIA336 appeared to be attenuated by 15-fold in BALB/c mice. Collectively, ApxI, but not other components in the exotoxin preparation of A. pleuropneumoniae serotype 10, was responsible for the hemolytic and cytotoxic effects on porcine erythrocytes and alveolar macrophages.     

Staphylococcus aureus and Escherichia coli elicit different innate immune responses from bovine mammary epithelial cells

Escherichia coli and Staphylococcus aureus are the most important pathogenic bacteria causing bovine clinical mastitis and subclinical mastitis, respectively. However, little is known about the molecular mechanisms underlying the different host response patterns caused by these bacteria. The aim of this study was to characterize the different innate immune responses of bovine mammary epithelium cells (MECs) to heat-inactivated E. coli and S. aureus. Gene expression of Toll-like receptor 2 (TLR2) and TLR4 was compared. The activation of nuclear factor kappa B (NF-κB) and the kinetics and levels of cytokine production were analyzed. The results show that the mRNA for TLR2 and TLR4 was up-regulated when the bovine MECs were stimulated with heat-inactivated E. coli, while only TLR2 mRNA was up-regulated when the bovine MECs were stimulated with heat-inactivated S. aureus. The expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and IL-8 increased more rapidly and higher when the bovine MECs were stimulated with heat-inactivated E. coli than when they were stimulated with heat-inactivated S. aureus. E. coli strongly activated NF-κB in the bovine MECs, while S. aureus failed to activate NF-κB. Heat-inactivated S. aureus could induce NF-κB activation when bovine MECs cultured in medium without fetal calf serum. These results were confirmed using TLR2- and TLR4/MD2-transfected HEK293 cells and suggested that differential TLR recognition and the lack of NF-κB activation account for the impaired immune response elicited by heat-inactivated S. aureus.     

Growth hormones therapy in immune response against Trypanosoma cruzi

Growth hormone (GH) is an important hypophyseal hormone that is primarily involved in body growth and metabolism. In mammals, control of Trypanosoma cruzi parasitism during the acute phase of infection is considered to be critically dependent on direct macrophage activation by cytokines. To explore the possibility that GH might be effective in the treatment of Chagas’ disease, we investigated its effects on the course of T. cruzi infection in rats, focusing our analyses on its influences on parasitemia, NO, TNF-α and IFN-γ concentration and on histopathological alterations and parasite burden in heart tissue. T. cruzi-infected male Wistar rats were intraperitoneally treated with 5 ng/10 g body weight/day of GH. Animals treated with GH showed a significant reduction in the number of blood trypomastigotes during the acute phase of infection compared with untreated animals (P < 0.05). For all experimental days (7, 14 and 21 post infection) of the acute phase, infected and GH treated animals reached higher concentrations of TNF-α, IFN-γ and nitric oxide as compared to untreated and infected counterparts (P < 0.05) Histopathological observations of heart tissue revealed that GH administration also resulted in fewer and smaller amastigote burdens, and less inflammatory infiltrate and tissue disorganization, indicating a reduced parasitism of this tissue. These results show that GH can be considered as an immunomodulator substance for controlling parasite replication and combined with the current drug used may represent in the future a new therapeutic tool to reduce the harmful effects of Chagas’ disease.     

Serum antibody responses in Creole kids experimentally infected with Haemonchus contortus

The objective of this study was to evaluate the relationship of parasite-specific serum antibodies with the resistance status of Creole kids. The average breeding values on egg output predicted in a context of natural infection at 11 months of age were distant of 1.07 genetic standard deviation between resistant and susceptible animals. After drenching the animals were maintained worm-free during 1 month until experimental infection with 10,000 Haemonchus contortus infective larvae (L3). Enzyme-linked immunosorbent assay was carried out in serum samples to determine the level of IgG, IgA and IgE anti-H. contortus L3 crude extracts and adult excretion/secretion products (ESP). Parasitological and blood immunological parameters were measured on the 2 extreme groups. Despite the absence of any typical signs of haemonchosis, susceptible kids had more than 11 times higher faecal egg counts (FEC) at 35 days post-infection (d.p.i.) than resistant kids had. Levels of immunoglobulin against H. contortus L3 and ESP increased significantly after infection in both groups. However, no difference in the host immune response mediated by immunoglobulin against H. contortus was evidenced between groups. This finding suggests that, in goats previously infected by H. contortus, a degree of protection occurred and the phenotypic and genetic segregation in resistant and susceptible animals were not related to the humoral immune response. The correlation coefficients between FEC and IgE anti-ESP (r=0.593; P<0.05 was significant in both resistant and susceptible animals. Such correlation suggesting a hypersensitivity reaction dependent on worm prolificacy has never been described. This result needs further studies to understand the mechanisms underlying this observation.     

Gene markers of generic Escherichia coli associated with colonization and persistence of Escherichia coli O157 in cattle

Enterohemorrhagic Escherichia coli (EHEC) O157 are important foodborne pathogens whose major reservoir are asymptomatic cattle. There is evidence suggesting that nonpathogenic E. coli and bacteriophages in the gastro-intestinal tract can influence the pathogenicity of EHEC O157. The factors contributing to the onset and persistence of shedding EHEC O157 in cattle are not completely elucidated. This study used Bayesian network analysis to identify genetic markers of generic E. coli associated with shedding of EHEC O157 in cattle from data generated during an oral experimental challenge study in 4 groups of 6 steers inoculated with three different EHEC O157 strains. The quantification of these associations was accomplished using mixed effects logistic regression. The results showed that the concurrent presence of generic E. coli carrying the prophage marker R4-N and the virulence marker stx2 increased the odds of the onset of EHEC O157 shedding. The presence of prophage markers z2322 and X011C increased, while C1.N decreased the odds of shedding EHEC O157 two days later. A significant antagonist interaction effect between the presence of the virulence marker stx2 on the day of shedding EHEC O157 and two days before shedding was also found. In terms of the persistence of EHEC O157 shedding, the presence of prophage marker R4-N (OR = 16, and 95% confidence interval (CI): 1.1, 252) was found to increase the odds of stopping EHEC O157 shedding, whereas prophage marker C1.N (OR = 0.16, CI: 0.03, 0.7) and the enterohemolysin gene hly (OR = 0.03, CI: 0.001, 0.8) were found to significantly decrease the odds of stopping EHEC O157 shedding. In conclusion, the study found that the presence of certain genetic markers in the generic E. coli genome can influence the pathogenicity of EHEC O157.     

Biofilm formation in Haemophilus parasuis: relationship with antibiotic resistance,serotype and genetic typing

Biofilms are surface-associated microbial communities, which are encased in self-synthesized extracellular environment. Biofilm formation may trigger drug resistance and inflammation, resulting in persistent infections. Haemophilus parasuis is the etiological agent of a systemic disease, Glässer's disease, characterized by fibrinous polyserositis, arthritis and meningitis in pigs. The purpose of this study was to examine the correlation between biofilm and antibiotic resistance among the clinical isolates of H. parasuis. In the present study, we tested biofilm-forming ability of 110 H. parasuis isolates from various farms using polystyrene microtiter plate assays. Seventy-three isolates of H. parasuis (66.4%) showed biofilm formation and most of them performed weak biofilm-forming ability (38/73). All isolates were tested for antimicrobial susceptibility to 18 antimicrobial agents by the broth microdilution method. H. parasuis isolates showed very high resistance (>90%) to sulfanilamide, nalidixic acid, and trimethoprim. Resistance to eight antibiotics such as penicillin (41.1% vs 8.1%), ampicillin (31.5% vs 8.1%), amoxicillin (28.8% vs 5.4%), gentamicin (46.6% vs 24.3%), cefazolin (19.2% vs 2.7%), doxycycline (19.2% vs 8.1%), cefotaxime (11% vs 2.7%), and cefaclor (13.7% vs 5.4%) was comparatively higher among biofilm producers than non-biofilm producers. Pulsed-field gel electrophoresis (PFGE) analyses could distinguish various isolates. Our data indicated that H. parasuis field isolates were able to form biofilms in vitro. In addition, biofilm positive strains had positive correlation with resistance to β-lactams antibiotics. Thus, biofilm formation may play important roles during H. parasuis infections.     

Rapid testing and quantification of Salmonella in ileocaecal lymph nodes of Austrian pigs slaughtered for consumption

Traditionally, quantitative microbial risk assessment (QMRA) is based on culture-dependent technologies. However, molecular quantification could forge additional, detailed information. A prerequisite of quantitative real-time PCR in animal science is a tissue preparation method where large volumes of tissue material can be reduced and particularly target cells can be concentrated. An easy-to-use sample preparation method for food (Matrix-Lysis) was recently adapted to tissues and now permits quantification of target cells from up to 5 g of organic matrix. The aim of this study was to examine the suitability of Matrix-Lysis for quantification of Salmonella in porcine ileocaecal lymph nodes (ICLNs). After demonstrating constant recovery rates, ICLNs from 540 pigs were examined for Salmonella spp. with Matrix-Lysis. Samples were also analysed using ISO 6579:2002, a combined enrichment/qPCR method and a lateral flow test. It could be shown that qPCR coupled with Matrix-Lysis can contribute to QMRA in food safety by enabling reproducible quantitative data, even at low contamination rates.     

MM3-ELISA evaluation of coproantigen release and serum antibody production in sheep experimentally infected with Fasciola hepatica and F. gigantica

During an experimental infection of sheep with Fasciola hepatica or F. gigantica, MM3-SERO and MM3-COPRO ELISA tests were applied to compare the kinetics of antibody production and coproantigen release between the 2nd and 32nd week post-infection (wpi). The Kato-Katz technique was used to measure the kinetics of egg shedding by both Fasciola species (eggs per gram of feces, epg). The kinetics of IgG antibodies for all sheep infected with F. hepatica and F. gigantica followed a similar pattern. Optical density (OD) increased rapidly between the 4th until the 12th wpi, when the highest values were reached and then decreased slowly until the 32nd wpi. Coproantigen levels increased above the cut-off value between 6 and 9 wpi in the F. hepatica group, and between 9 and 11 wpi in the F. gigantica group. The comparison between coproantigen levels and epg indicated that F. hepatica-infected sheep had detectable amounts of coproantigens 4–7 weeks before patency (egg shedding), while F. gigantica-infected sheep had detectable amounts of coproantigens 3–6 weeks before patency. When comparing the kinetics of coproantigen release vs the kinetics of epg, a similar pattern emerged, but with a two-week time-lag in epg, for both F. hepatica and F. gigantica infections. The amount of coproantigen release by each adult was not burden dependent for F. hepatica infection (burden of 33–66 adults), while it was for F. gigantica infection (burden of 17–69 adults). The results demonstrate the usefulness of the MM3-SERO and MM3-COPRO ELISAs as tools for the diagnosis of early as well as long-term fascioliasis infections, and suggest that they can potentially be applied to human fascioliasis even in countries where F. hepatica and F. gigantica co-exist. These tests can be employed not only in the diagnosis, but also in studies on epidemiology as well as pathogenesis and treatment in animals and humans since they allow post-treatment infection monitoring.     

Isolation and characterization of a bovine isolate of Neospora caninum with low virulence

Neospora caninum tachyzoites were isolated from the brain of an asymptomatic naturally infected calf with precolostral-specific antibodies. The new isolate, named Nc-Spain 1H, was identified as a member of the N. caninum species based on its internal transcribed spacer 1 (ITS-1) sequence and was genetically characterized using microsatellite markers. Multilocus analysis showed that Nc-Spain 1H was genetically different from other N. caninum isolates. We compared the in vitro tachyzoite yield and viability rate of the Nc-Spain 1H and Nc-1 isolates in a plaque assay. The lower tachyzoite yields displayed by Nc-Spain 1H were complemented with a significantly lower viability rate. Moreover, in an in vitro tachyzoite–bradyzoite stage conversion assay, the percentage of Nc-Spain 1H bradyzoite conversion was similar to that of the cystogenic isolate Nc-Liv, with the exception that Nc-Spain 1H produced only intermediate bradyzoites. The pathogenicity of Nc-Spain 1H was examined in BALB/c mice, and the results demonstrated that Nc-Spain 1H failed to induce clinical signs or mortality and that no parasite DNA was detected in the brain during the chronic stage of infection. In a pregnant mouse model, Nc-1 infection resulted in high transplacental transmission, leading to a high neonatal mortality rate over time. In contrast, the offspring survival rate from Nc-Spain 1H-infected dams was almost 100%, and N. caninum DNA was detected in only one pup. These data show that Nc-Spain 1H appears to be a low virulence isolate and may be a suitable candidate for live vaccine development.