Increase of Diversity of Mumps Virus Genotype G SH Variants Circulating Among a Highly Immunized Population: Spain, 2007-2019.

MuV caused three epidemic waves in Spain since genotype G emerged in 2005, despite high vaccination coverage. SH gene sequencing according to WHO protocols allowed the identification of seven relevant variants and 88 haplotypes. While the originally imported MuVi/Sheffield.GBR/1.05/-variant prevailed during the first two waves, it was subsequently replaced by other variants originated by either local evolution or importation, according to the additional analysis of hypervariable NCRs. The time of emergence of the MRCA of each MuV variant clade was concordant with the data of the earliest sequence. The analysis of Shannon entropy showed an accumulation of variability on six particular positions as the cause of the increase on the number of circulating SH variants. Consequently, SH gene sequencing needs to be complemented with other more variable markers for mumps surveillance immediately after the emergence of a new genotype, but the subsequent emergence of new SH variants turns it unnecessary.

[Rapid spread of the HIV-1 circular recombinant CRF02-AG in Russia and neighboring countries].

The spread of the HIV-1circular recombinant CRF02-AG in countries of the former Soviet union (Commonwealth of Independent States, CIS) was studies using partial and full genome sequences. The full-genome sequence of the CRF02-AG recombinant circulating in Russia was obtained for the first time. A Global phylogenetic tree of CRF02-AG full-genome sequences was constructed. Three distinct groups of the sequences were detected as clustered by the geographical location (CIS, South Korea, and France), which is indicative of the single-virus introduction in each of the regions mentioned above. The CIS cluster exhibiting minimum genetic diversity was, therefore, relatively young. The phylogenetic analysis of the env gene sequences within the CIS cluster made it possible to clearly discriminate three branches: two of Russian and one of Uzbek origin. The low genetic diversity within the two Russian subclusters provides evidence of at least two recent independent introductions of the CRF02-AG recombinant from Central Asia into Russia. This work was performed within the framework of the 7th Federal Research Program (FP&), Project EURIPRED (European Research Infrastructures for Poverty Related Diseases), grant agreement No.312661.

Controversies in point-of-care 3D printing for oncological and reconstructive surgery with free software in oral and maxillofacial surgery: European regulations, costs, and timeframe.

The aim of this paper is to discuss the controversies surrounding the most recent European regulations, as well as the cost, for a 3D printing workflow using free-source software in the context of a tertiary level university hospital in the Spanish public health system. Computer-aided design and manufacturing (CAD/CAM) for head and neck oncological surgery with the printing of biomodels, cutting guides, and patient-specific implants has made it possible to simplify and make this type of highly complex surgery more predictable. This technology is not without drawbacks, such as increased costs and the lead times when planning with the biomedical industry. A review of the current European legislation and the literature on this subject was performed, and comparisons made with the authors' in-house 3D printing setup using free software and different 3D printers. The cost analysis revealed that for the cheapest setup with free software, it would be possible to amortize the investment from case 2, and in all cases the initial investment would be amortized before case 9. The timeframe ranged from 2 weeks with the biomedical industry to 72 h with point-of-care 3D printing. It is now possible to develop point-of-care 3D printing in any hospital with almost any budget.

Correlation between the main clinical, imaging, and arthroscopy findings in patients with temporomandibular disorders.

The purpose of this work was to prospectively correlate the most characteristic clinical symptoms of temporomandibular disorders, such as pain and limitation of mouth opening, with the findings of magnetic resonance imaging (disc position, degenerative changes, and effusion) and arthroscopy findings (roofing, synovitis, chondromalacia, adhesions, and perforations). These examinations were performed in 298 patients diagnosed with internal derangement refractory to conservative treatment. The mean age of the patients was 38.59 years; 92.6% were female. The t-test and one-way analysis of variance (ANOVA) were used to correlate the findings. Significant relationships were found between pain and disc displacement without reduction (P = 0.033) and effusion (P = 0.003) on MRI, coinciding with correlations between pain and roofing of 0-25% (P = 0.016) and synovitis (P = 0.001) on arthroscopy. A significant relationship was also observed between mouth opening limitation and the presence of osteoarthrosis (P = 0.018) on MRI, and between mouth opening limitation and synovitis (P = 0.022), chondromalacia (P = 0.002), and adhesions (P < 0.001) on arthroscopy. All of these findings were observed in patients with a poor initial clinical situation, which highlights the considerable potential of correlating these data with imaging and arthroscopy findings.

Benchmarking in the process of donation after brain death: a methodology to identify best performer hospitals.

A benchmarking approach was developed in Spain to identify and spread critical success factors in the process of donation after brain death. This paper describes the methodology to identify the best performer hospitals in the period 2003-2007 with 106 hospitals throughout the country participating in the project. The process of donation after brain death was structured into three phases: referral of possible donors after brain death (DBD) to critical care units (CCUs) from outside units, management of possible DBDs within the CCUs and obtaining consent for organ donation. Indicators to assess performance in each phase were constructed and the factors influencing these indicators were studied to ensure that comparable groups of hospitals could be established. Availability of neurosurgery and CCU resources had a positive impact on the referral of possible DBDs to CCUs and those hospitals with fewer annual potential DBDs more frequently achieved 100% consent rates. Hospitals were grouped into each subprocess according to influencing factors. Hospitals with the best results were identified for each phase and hospital group. The subsequent study of their practices will lead to the identification of critical factors for success, which implemented in an adapted way should fortunately lead to increasing organ availability.

Hospitalizations due to and with chronic obstructive pulmonary disease in Galicia: 20 years of evolution.

OBJECTIVE: This work aims to analyze the evolution of COPD-related hospitalizations in Galicia from 1996 to 2018 both as main cause of admission (DUE TO COPD) or when coded in any diagnostic order (WITH COPD), assessing the influence of age, sex, seasonality, and other main causes of the hospitalization. METHODS: An analysis was conducted of administrative healthcare database (CMBD) data on index COPD-related hospitalizations in Galicia from 1996 to 2018. Crude, specific, and standardized rates were calculated for the entire sample and according to age and sex groups. RESULTS: In the period from 1996 to 2018, there were 310,883 index admissions WITH COPD in Galicia, of which 29.6% were DUE TO COPD. Both WITH COPD and DUE TO COPD admission rates increased, mainly in men. There was a clear seasonality that was especially relevant in the DUE TO COPD group. The mean male-to-female ratio was approximately 4:1 in both groups, although it was lower in the extreme age groups, with no change over time. The mean age at admission increased three years in men during this period; there were no changes among women. The main causes of admission in those not hospitalized DUE TO COPD were heart failure and pneumonia. CONCLUSION: The combined evaluation of records of hospital admissions WITH COPD and DUE TO COPD offers additional information for a better understanding of the trends of this disease and allows for establishing hypotheses that explain the results described, providing information for better healthcare planning.

Identification of a gene cluster for cell-surface genes of the SRS superfamily in Neospora caninum and characterization of the novel SRS9 gene.

Here we present the detection of a gene cluster for Neospora caninum surface genes, similar to the Toxoplasma gondii SRS9 locus, and the cloning and characterization of the NcSRS9 gene. PCR genome walking, using NcBSR4 gene as a framework, allows the identification, upstream NcBSR4, of 2 sequences homologous to the SRS5 and the Ubiquinol-cytochrome C reductase genes and, downstream NcBSR4, of an ORF of 1191 bp coding for a 396-amino acid polypeptide with 59% similarity to the TgSRS9 antigen. A putative 39-residue signal peptide was found at the NH2-terminus followed by a hydrophilic region, and a potential site for a glycosylphosphatidylinositol anchor at the COOH-terminus. A recombinant NcSRS9 protein was produced and was recognized on a Western blot by a low proportion of sera from a panel of naturally infected cows and calves. In addition, Western blot analysis using polyclonal anti-rNcSRS9 revealed stage-specific expression of NcSRS9 in bradyzoites but not in tachyzoites, and immunohistochemistry on brain from a congenitally infected calf showed NcSRS9 recognition in bradyzoites contained in tissue cysts. However, bradyzoite-specific expression of NcSRS9 could not be proven by immunofluorescence on bradyzoites obtained in vitro and RT-PCR analysis showed no significant variations of NcSRS9 transcripts during in vitro tachyzoite-bradyzoite switch, probably due to incomplete maturity of in vitro bradyzoites. Initial characterization of NcSRS9 in this study may lead to further studies for a better understanding of N. caninum persistence.

Smart contact lens: a promising therapeutic tool in aniridia. / Lente de contacto inteligente: una prometedora herramienta terapéutica en aniridia.

OBJECTIVE: To perform pre-clinical testing using optical design tools to simulate the optical quality of a smart artificial iris platform encapsulated in a scleral contact lens. These tools allow us to generate aniridia eye models and evaluate different metrics of visual quality and retinal illumination based on the aperture of the artificial iris based on liquid crystals. METHOD: The OCT imaging technique was used to measure the geometry of the anterior segment in a patient with aniridia and, from these data, the eye model was generated with the Zemax optical design program and specific programs developed in Matlab. Ocular aberrations were calculated and the visual function of the anirida eye model was evaluated in three scenarios: (i) without optical correction, (ii) with correction with a commercial scleral contact lens, and (iii) with correction with an optical lens. intelligent contact based on artificial iris. RESULTS: Optical quality in patients with aniridia is limited by the magnitude of high-order aberrations. Conventional scleral contact lens design accurately corrects for blur but is unable to compensate for high-order ocular aberrations, especially spherical aberrations. The artificial iris-based smart contact lens design enables virtually all high-order aberrations to be compensated with active control of the pupillary diameter (activation of liquid crystal cells based on ambient lighting). In addition to minimizing high-order aberrations, reducing the pupil size would increase the depth of focus. CONCLUSIONS: This article demonstrates by means of optical simulations the concept of an intelligent artificial iris platform encapsulated in a scleral contact lens and its possible application in patients with aniridia. Furthermore, it allows us to anticipate possible visual results in clinical trials with healthy patients (after application of mydriatic agents) and in patients with aniridia. The results demonstrate a better visual quality and a decrease in retinal illumination.

Smart contact lens: A promising therapeutic tool in aniridia.

OBJECTIVE: The perform pre-clinical testing using optical design tools to simulate the optical quality of a smart artificial iris platform encapsulated in a scleral contact lens. These tools allow us to generate aniridia eye models and evaluate different metrics of visual quality and retinal illumination based on the aperture of the artificial iris based on liquid crystals. METHOD: The OCT imaging technique was used to measure the geometry of the anterior segment in a patient with aniridia and, from these data, the eye model was generated with the Zemax optical design program and specific programs developed in Matlab. Ocular aberrations were calculated and the visual function of the anirida eye model was evaluated in three scenarios: (i) without optical correction, (ii) with correction with a commercial scleral contact lens, and (iii) with correction with an optical lens. Intelligent contact based on artificial iris. RESULTS: Optical quality in patients with aniridia is limited by the magnitude of high-order aberrations. Conventional scleral contact lens design accurately corrects for blur but is unable to compensate for high-order ocular aberrations, especially spherical aberrations. The artificial iris-based smart contact lens design enables virtually all high-order aberrations to be compensated with active control of the pupillary diameter (activation of liquid crystal cells based on ambient lighting). In addition to minimizing high-order aberrations, reducing the pupil size would increase the depth of focus. CONCLUSIONS: This article demonstrates by means of optical simulations the concept of an intelligent artificial iris platform encapsulated in a scleral contact lens and its possible application in patients with aniridia. Furthermore, it allows us to anticipate possible visual results in clinical trials with healthy patients (after application of mydriatic agents) and in patients with aniridia. The results demonstrate a better visual quality and a decrease in retinal illumination.

Stage-specific expression of Nc SAG4 as a marker of chronic Neospora caninum infection in a mouse model.

Neospora caninum infection persists throughout the life of its intermediate host due to the conversion of tachyzoites to slowly dividing bradyzoites that encyst in the brain. This event results in persistent N. caninum infection in bovine herds and partially explains the poor efficacy of many chemotherapeutic agents and vaccine formulations. Thus, there is a need for greater understanding of the tachyzoite-to-bradyzoite conversion mechanisms. Here we studied for the first time the transcription kinetics of the N. caninum bradyzoite-specific gene NcSAG4 in brain samples from chronically infected mice by means of real-time RT-PCR. NcSAG4-messenger RNA (mRNA) levels increased significantly during the chronic phase but followed 2 different expression patterns depending on the isolate used for murine inoculation. NcSAG4-mRNA levels in brains from Nc-1-inoculated mice peaked during late chronic infection (on day 64 post-infection, p.i.), whereas those from Nc-Liv-inoculated mice peaked earlier during the chronic infection (on day 32 p.i.). This difference could be a reflection of the different abilities of these isolates to replicate and form cysts in parasitized brains. These results are consistent with our observations of anti-rNcSAG4 antibody production; low levels were present at seroconversion and slowly increased during the chronic phase. In contrast, NcSAG1 transcription levels, which mark the tachyzoite stage, were maintained without variation in both groups of mice. This suggests the presence of a significant amount of tachyzoites or intermediate zoites expressing NcSAG1 in the brain, even during the late chronic infection.

First isolation of Besnoitia besnoiti from a chronically infected cow in Spain.

Besnoitia besnoiti was isolated from a skin biopsy of a chronically infected cow from central Spain. Zoites released from macroscopic cysts were adapted to its culture in vitro on a MARC-145 cell monolayer. Tachyzoites produced in vitro were either cryopreserved or used for genomic DNA isolation. A 2206 nt sequence containing 18S ribosomal RNA gene, internal transcribed spacer 1 (ITS 1), and a partial sequence of 5.8S ribosomal RNA gene was amplified by PCR and sequenced. This sequence showed a 99-100% identity to 18S, ITS1, and 5.8S sequences of B. besnoiti published in databases. After analysis by transmission and scanning electron microscopy of isolated bradyzoites and tachyzoites, it was observed that their ultrastructural morphology coincided with B. besnoiti. The isolate characterized in this study was identified as B. besnoiti on the basis of the disease produced, molecular characteristics, and morphology. The B. besnoiti isolate was denoted as BbSpain-1; it is the first isolate obtained and characterized in Spain and one of the first European isolates adapted to grow in vitro. The isolation and in vitro production of this B. besnoiti isolate offers a good opportunity to study general aspects of bovine besnoitiosis, including epidemiology, pathogenesis, and diagnosis of this re-emergent disease.

Incidence of non-B subtypes of HIV-1 in Galicia, Spain: high frequency and diversity of HIV-1 among men who have sex with men.

An increase in HIV transmission among men who have sex with men (MSM) has been reported in eight regions of Spain from 2003 to 2007. In order to study the incidence of HIV-1 genetic forms in Galicia, northwest of Spain, in particular the spread of HIV-1 variants among MSM, 93 newly diagnosed HIV-1 patients, including those with acute and recently acquired infections, were studied for a year from August 2008 to August 2009. Thirty eight (41%) were MSM. Of them, nine (24%) were infected by non-B viruses, including seven different genetic forms. The analysis of transmission clusters showed that 23 (60%) MSM grouped in different clusters and mostly in large clusters. Resistance mutations were detected in six (16%) MSM.

Review of catastrophic hand diagnosis over 15 years in a tertiary hospital: do we make proper use of the term? / Revisión del diagnóstico de mano catastrófica a lo largo de 15 años en un hospital terciario: ¿hacemos un uso adecuado del término?

OBJECTIVE: To describe the characteristics of patients diagnosed with 'catastrophic hand' by the plastic surgery department in a tertiary hospital in the period between 2000-2015, analyse the evolution of the use of the term 'catastrophic hand' and conduct a review of the literature with a view to proposing the optimal definition for said expression. MATERIAL AND METHOD: We conducted a descriptive and retrospective study of patients diagnosed with 'catastrophic hand' who required hospital care by the plastic surgery department of a tertiary hospital in the period between 2000-2015. We conducted a literature review on the use of the term 'catastrophic hand' and we applied the classifications proposed in the publications consulted to our results. RESULTS: The number of 'catastrophic hand' diagnoses was 133. We observed a downward trend in the use of the term over the years. Applying classifications based on the impossibility of recovery of an acceptable hand (presence of three long fingers and thumb) by reconstructive procedures, only 9 out of 133 patients could be considered to have 'catastrophic hands', constituting 6.7% of the total cases. DISCUSSION: The term 'catastrophic hand' has different meanings, and this generates ambiguity. The use of a classification based on the possibility of obtaining an acceptable hand diminishes its use and improves the approach for such injuries. CONCLUSION: We advocate limiting the use of 'catastrophic hand' to cases that meet criteria of 'mutilated hand' and 'metacarpal hand' to avoid indiscriminate use of the term and optimise therapeutic management.

Isolation and genetic characterization of Neospora caninum from asymptomatic calves in Spain.

Neospora caninum is a cyst-forming parasite that causes abortion in cattle. Despite this parasite's ubiquitous distribution and wide host range, the number of N. caninum isolates obtained to date is limited. In vitro isolation of the parasite is arduous and often unsuccessful. In addition, most isolates have been obtained from clinically affected hosts and therefore could be biased towards more virulent isolates. In this report, an improved isolation approach from transplacentally infected newborn calves was undertaken and 9 new isolates were obtained. Moreover, a microsatellite technique was applied to investigate the genetic diversity of these isolates. Most isolates showed specific genetic profiles. However, the Nc-Spain10 isolate was identical to the previously described Nc-Spain1H isolate and Nc-Spain3H was identical to Nc-Spain4H. These isolates were likely to have identical genotypes because they were isolated from distinct calves of the same herd. Future pathogenic characterization of these isolates will contribute to the investigation of the relationship between isolate virulence and the outcome of infection, as well as other epidemiological features, such as transmission.

Usefulness of rNcGRA7- and rNcSAG4-based ELISA tests for distinguishing primo-infection, recrudescence, and chronic bovine neosporosis.

Bovine reproductive failure caused by the parasite Neospora caninum is a major problem and is responsible for severe economic losses worldwide. Currently, appropriate control measures depend on the predominant transmission route in a particular herd. Therefore, the development of diagnostic tools capable of discriminating between primo-infection, recrudescence, re-infection, and chronic infection is a major challenge in the serodiagnosis of bovine neosporosis. Here, two recombinant protein-based ELISAs utilizing the immunodominant NcGRA7 dense granule protein and the NcSAG4 bradyzoite stage-specific protein were developed and showed good diagnostic performances. Their usefulness for discerning between primo-infection, recrudescence, re-infection, and chronic infection was also studied by analyzing an appropriate panel of serum samples belonging to different groups of experimentally and naturally infected bovines. Our results suggest that anti-rNcGRA7 antibody levels may be indicative of acute infection (primo-infection, re-infection, and recrudescence), whereas the presence of anti-rNcSAG4 antibodies may be associated with chronic infection and could be a good indicator of infection establishment (tachyzoite-bradyzoite conversion). Moreover, primo-infection associated with a Neospora-associated epidemic abortion pattern is characterized by the detection of anti-rNcGRA7 antibodies together with the absence or detection of anti-rNcSAG4 antibody levels around the cut-off point. In contrast, the detection of antibody levels directed against both recombinant proteins may be quite indicative of recrudescence or re-infection associated with abortion and/or vertical transmission in herds with a Neospora-associated endemic abortion pattern. In conclusion, both serological tests developed in the present study offer additional information to conventional avidity tests and, consequently, improve the diagnosis of bovine neosporosis with perspectives for control measures.

Molecular characterisation of BSR4, a novel bradyzoite-specific gene from Neospora caninum.

Here we present the identification and cloning of the NcBSR4 gene, the putative Neospora caninum orthologue to the Toxoplasma gondii TgBSR4 gene. To isolate NcBSR4, genome walking PCR was performed on N. caninum genomic DNA using the expressed sequence tag NcEST3c28h02.y1 sequence, which shares a 44% identity with the TgBSR4 gene, as a framework. Nucleotide sequencing of amplified DNA fragments revealed a single uninterrupted 1227 bp open reading frame that encodes a protein of 408 amino acids with 66% similarity to the TgBSR4 antigen. A putative 39-residue signal peptide was found at the NH2-terminus, followed by a hydrophilic region. At the COOH-terminus, a potential site for a glycosylphosphatidylinositol anchor was identified at amino acid 379. A polyclonal serum against recombinant NcBSR4 protein was raised in rabbits, and immunolabelling demonstrated stage-specific expression of the NcBSR4 antigen in N. caninum bradyzoites produced in vitro and in vivo. Furthermore, RT-PCR analysis showed a slight increase of NcBSR4 transcripts in bradyzoites generated during in vitro tachyzoite-to-bradyzoite stage-conversion, suggesting that this gene is specifically expressed at the bradyzoite stage and that its transcription relies on the switch to this stage.

Glyoxal and methylglyoxal as urinary markers of diabetes. Determination using a dispersive liquid-liquid microextraction procedure combined with gas chromatography-mass spectrometry.

Glyoxal (GO) and methylglyoxal (MGO) are α-oxoaldehydes that can be used as urinary diabetes markers. In this study, their levels were measured using a sample preparation procedure based on salting-out assisted liquid-liquid extraction (SALLE) and dispersive liquid-liquid microextraction (DLLME) combined with gas chromatography-mass spectrometry (GC-MS). The effect of the derivatization reaction with 2,3-diaminonaphthalene, the addition of acetonitrile and sodium chloride to urine, and the DLLME step using the acetonitrile extract as dispersant solvent and carbon tetrachloride as extractant solvent were carefully optimized. Quantification was performed by the internal standard method, using 5-bromo-2-chloroanisole. The intraday and interday precisions were lower than 6%. Limits of detection were 0.12 and 0.06ngmL-1, and enrichment factors 140 and 130 for GO and MGO, respectively. The concentrations of these α-oxoaldehydes in urine were between 0.9 and 35.8ngg-1 levels (creatinine adjusted). A statistical comparison of the analyte contents of urine samples from non-diabetic and diabetic patients pointed to significant differences (P=0.046, 24 subjects investigated), particularly regarding MGO, which was higher in diabetic patients. The novelty of this study compared with previous procedures lies in the treatment of the urine sample by SALLE based on the addition of acetonitrile and sodium chloride to the urine. The DLLME procedure is performed with a sedimented drop of the extractant solvent, without a surfactant reagent, and using acetonitrile as dispersant solvent. Separation of the analytes was performed using GC-MS detection, being the analytes unequivocal identified. The proposed procedure is the first microextraction method applied to the analysis of urine samples from diabetic and non-diabetic patients that allows a clear differentiation between both groups using a simple analysis.

Identification and molecular cloning of the Neospora caninum SAG4 gene specifically expressed at bradyzoite stage.

Here, we identify and clone the NcSAG4 gene, orthologue to the Toxoplasma gondii TgSAG4 gene, and the first reported gene to be expressed specifically during the Neospora caninum bradyzoite stage. To isolate NcSAG4, we designed degenerate oligonucleotides based on the TgSAG4 protein amino acid sequence. A 312-bp DNA fragment was amplified by PCR from N. caninum genomic DNA, whose sequence showed 65% identity to TgSAG4 gene over 257 bp. NcSAG4 gene sequence was obtained by PCR genome walking. Nucleotide sequencing of amplified DNA fragments showed a single uninterrupted 522-bp ORF that encoded a 173-amino-acid protein with a predicted molecular mass of 18,394 Da, with 69% similarity to the TgSAG4 antigen. A 28-residue putative signal peptide was found at the NH2-terminus, followed by a strongly hydrophilic region. An amino acid motif for a phosphatidylinositol glycan anchor was identified at the COOH-terminus. The NcSAG4 protein lacking the putative signal peptide at the NH2-terminus was expressed in Escherichia coli and was recognized in western blot by sera from congenitally infected cattle. A mouse polyclonal anti-rNcSAG4 serum was produced for immunofluorescence studies, and revealed stage-specific NcSAG4 antigen expression in in vitro-cultured bradyzoites. Real-time reverse transcription-PCR analysis with samples from in vitro stage-conversion assay showed increasing levels of NcSAG4 transcript over time, suggesting a developmental upregulation of this gene.

Comparison of Neospora caninum distribution, parasite loads and lesions between epidemic and endemic bovine abortion cases.

It has been suggested that the abortion herd pattern could influence bovine foetal neosporosis. Here, a comparison of (i) Neospora caninum DNA-detectability by PCR, (ii) N. caninum-associated lesions and (iii) parasite loads in target organs was made between epidemic and endemic abortion cases. We observed that N. caninum DNA was predominantly detected in more than one organ in the foetuses from herds with epizootic rather than endemic abortion cases (P<0.05, Fisher F-test). The highest parasite burdens were found in the heart in foetuses from outbreaks of epidemic abortion and in the brain in endemic cases (P<0.05, Kruskal-Wallis H-test). Moreover, foetuses from epidemic outbreaks had significantly higher parasite burdens in heart (P<0.05, Mann-Whitney U-test) than endemic abortion cases. Epidemic abortion cases showed higher lesion frequencies in liver (P<0.05, Fisher F-test). This report confirms that the abortion herd pattern is an important factor that influences pathogenesis in natural N. caninum infections.