Sequence and phylogenetic analysis of the 16S rRNA gene of Ehrlichia canis strains in dogs with clinical monocytic ehrlichiosis

The purpose of this study was to characterize, at the molecular level, the Ehrlichia canis strains involved in naturally occurring canine monocytic ehrlichiosis (CME) in Greece, and to investigate if any sequence diversity exists between the 16S rRNA genes of those involved in the mild non-myelosuppressive or the severe myelosuppressive form of CME. To this end, amplification of the ehrlichial 16S rRNA gene was attempted by nested polymerase chain reaction (PCR) assays in bone marrow (BM) aspirates from 20 dogs tentatively diagnosed as having non-myelosuppressive (n=10, group A) or myelosuppressive (n=10, group B) CME. PCR assay using E. canis-specific primers revealed that 15 BM samples, including all group A and 5 group B dogs, were positive. Using universal PCR primers, a nearly full-length 16S rRNA gene could be amplified from 13 BM samples, including 9 group A and 4 group B dogs. The 16S rDNA analysis based on secondary structure revealed that all sequences of the Greek strains were identical to each other and indicated 100% identity among some American (Venezuelan and Brazilian), European (Greek), Middle Eastern (Turkish) and Asiatic (Thailand) strains. The results of this study suggest that the E. canis strains involved in the non-myelosuppressive and myelosuppressive forms of CME in Greece share an identical 16S rRNA genotype.     

An Analysis of Urban Size and Territorial Location Effects on Employment Probabilities: The Spanish Case

The probability of being employed varies depending on several factors. Many of these are related to personal characteristics such as educational level, age, gender, or number and age of children. Nevertheless, other factors may be relevant, in particular the geographical environment. The purpose of this paper is to analyse the relevance of urban size and the position of each territory (in terms of its distance from large metropolises) for the probability of being employed in the Spanish economy. Following the set of economic regions suggested by Polése, Shearmur and Rubiera (2007 ), we try to explain the spatial patterns of employment distribution. Our results show some relevant differences between these alternative economic areas. We find that municipalities with similar sizes and located at a similar distance from a metropolis but belonging to different Autonomous Communities or provinces share similar employability patterns.     

Experimental infection of pregnant ewes with enteric and abortion-source Chlamydophila abortus

Two groups of pregnant ewes were experimentally infected oronasally in midpregnancy. A faecal and an abortion-source isolate of Chlamydophila abortus were used. They were derived from a healthy ewe from a flock with no history of abortion, and from an aborted foetus in a farm with enzootic abortion. As assessed by modified Ziehl-Neelsen (MZN) staining, egg culture, antigen ELISA, the Clearview test and immunohistochemistry, inoculation resulted in placental and/or foetal infection in all ewes. Histopathology revealed placentitis in two and four ewes inoculated with the enteric or abortion-source isolate, respectively, in addition, these samples were immunohistochemically positive for chlamydial antigen. All six ewes infected with the enteric isolate and five of seven ewes infected with the abortion-source isolate showed evidence for a serological response by an indirect ELISA or CFT. Neither chlamydiae nor lesions were detected in the placentae and lambs of the uninfected control ewes, which remained seronegative. Our results suggest that enteric C. abortus can be associated with placental and foetal lesions in sheep.     

Evaluation of a serum-based PCR assay for the diagnosis of canine monocytic ehrlichiosis

The aim of this study was to estimate the relative diagnostic sensitivity and specificity of a polymerase chain reaction (PCR) assay in the serum of dogs with naturally occurring non-myelosuppressive canine monocytic ehrlichiosis (CME), and to investigate the association between PCR positivity and immunofluorescence antibody (IFA) titres for Ehrlichia canis. Serum samples obtained from 38 dogs with non-myelosuppressive CME and 12 healthy dogs were analyzed retrospectively. Each serum sample was analyzed in triplicate using an E. canis-specific nested PCR assay targeting a 389 bp sequence of the 16S rRNA gene. E. canis DNA was amplified in 24 of 38 (63.1%) affected dogs; all samples from healthy dogs were negative. A high level of agreement was found among the PCR replicates (P < 0.0001). Median IFA titre of the 24 PCR-positive dogs was significantly lower than that of the PCR-negative infected dogs (P = 0.0029), indicating that E. canis DNA may circulate prior to the development of a high antibody titre. Serum-based PCR analysis is suggested for the early diagnosis of CME when whole blood samples are not available.     

Pharmacodynamics of amoxicillin against Mannheimia haemolytica and Pasteurella multocida and pharmacokinetic/pharmacodynamic (PK/PD) correlation in sheep

Silicone-made tissue cages were implanted in sheep. Blood serum (SBS) and tissue cage fluid (TCF) samples were collected after amoxicillin intravenous and intramuscular administrations, at the dose of 15 mg/kg. Amoxicillin pharmacodynamics were studied in an artificial culture medium, SBS and TCF with use of a Mannheimia haemolytica and a Pasteurella multocida strain. A concentration-independent antimicrobial activity of amoxicillin was confirmed for levels higher than 0.79–1.75 × MIC. This result favored the use of the percentage of the 24 h dosing interval during which drug levels remain above MIC as the appropriate pharmacokinetic/pharmacodynamic index. The subsequent correlation revealed that intravenous administration could be considered effective against “deep” infections caused by bacteria with MICs < 1 μg/mL or “shallow” infections caused by bacteria with MICs < 0.1 μg/mL. Intramuscular administration could be safely considered effective against both “deep” and “shallow” infections when the MICs of the targeted pathogens are lower than 1 μg/mL.     

Selenium and vitamin E effect on antibody production of sheep vaccinated against enzootic abortion (Chlamydia psittaci)

The effect of selenium (Se) and vitamin E (vit E) on antibody production of sheep vaccinated against Chlamydia psittaci (ovis) was investigated. Thirty-two sheep, one year old, seronegative to Chlamydia infection, vaccinated against enterotoxemia and dewormed were used. Injectable sodium selenite (0.1 mg/kg b.w.) was given twice to animals of the first group (gSe), with a three week interval. The sheep of the second group (gE) received 1 g vit E each orally, six times at weekly intervals. The animals of the third group (gSeE) were given Se and vit E in doses and routes of administration as in gSe and gE. The animals of the fourth group served as controls (gC) and injected normal saline. The first vaccination was made at the time that the second Se injection was given. Revaccination was made two weeks later. The experiment lasted 29 weeks. The results indicated that Se alone led to a significant increase of Chlamydia antibody response (P < 0.05), but not when it was given in combination with vit E. Animals that received vit E (gE) had much lower titres, just above of those of the controls.     

Serum acute phase proteins as clinical phase indicators and outcome predictors in naturally occurring canine monocytic ehrlichiosis

Background: Canine monocytic ehrlichiosis (CME), caused by Ehrlichia canis, is an important tick‐borne disease of global importance. Currently, limited information is available on the diagnostic and prognostic value of acute phase proteins (APPs) in dogs naturally infected with E. canis. Hypothesis: APPs may be useful indicators of the clinical phase of CME and predictive of the clinical outcome (death or survival). Animals: Fifty‐six dogs naturally infected with E. canis and 7 clinically healthy control dogs. Methods: C‐reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp), and albumin concentrations determined on admission were retrospectively compared among 27 dogs with nonmyelosuppressive CME, 29 dogs with myelosuppressive CME and 7 healthy dogs. Diagnosis of CME was based on clinical and clinicopathological findings, seropositivity to E. canis, polymerase chain reaction amplification of E. canis‐specific 16S rDNA, microscopic observation of Ehrlichia sp. morulae in blood monocytes or some combination of these. Results: Mean concentrations of CRP, SAA, and Hp were significantly higher in the myelosuppressed dogs compared with the other groups, but no significant differences were found in the concentration of albumin. Survival analysis of the affected animals indicated that APP concentrations were not associated with clinical outcome; the latter was strongly associated with pancytopenia (odds ratio for death 22.7) and neutropenia (odds ratio for death 7.7). Conclusions and Clinical Importance: CRP, SAA, and Hp serum concentrations on admission are useful indicators of the clinical phase of CME, but are not useful predictors of clinical outcome.     

Subspecies variation in Greek strains of Chlamydophila abortus

The Greek chlamydial strains FAS, FAG, VPG and LLG, isolated from aborted sheep or goat foetuses, had been previously characterized as divergent on the basis of mouse cross-protection experiments, with LLG and its homologous POS significantly different from the rest in inclusion morphology, polypeptide profiles and reactivity with monoclonal antibodies. To determine the genetic basis of their divergence the 16S-23S ribosomal intergenic spacer was analysed by RFLP analysis of PCR 16SF2/23R amplicons. Using the restriction enzymes BfaI, SfcI, HpaI, BclI, DdeI and AclI, the strains were classified as Chlamydophila abortus. However, digestion with RsaI made it possible to differentiate strains FAS, FAG and VPG from strains LLG and POS, generating DNA fragments of 530/55 and 585bp, respectively. By subsequent sequence analysis of the 23S domain I rRNA gene only strain FAS was identical to reference strain A22 of C. abortus. Strains FAG and VPG presented an identical nucleotide deviation at position 593 of signature sequences. Strains LLG and POS presented three identical nucleotide deviations at positions 156, 186 and 307. Variation within the domain I signature sequences for the examined abortion strains was < or =0.69%. In conclusion, substantial genetic and biological diversity among strains of C. abortus was demonstrated, suggesting that subspecies variation status for certain strains may be applicable. Our findings suggest that differentiation may be possible at a subspecies level by RFLP analysis.     

Cytologic patterns of lymphadenopathy in canine monocytic ehrlichiosis

Background: Recognition of different cytologic patterns in lymph nodes (LNs) from dogs with canine monocytic ehrlichiosis (CME) and noninfectious causes of lymphoid reactivity may have diagnostic utility. Objectives: The aims of the present study were to compare cytologic patterns in LNs of dogs with different phases of CME, to investigate the association of cytologic pattern and presence of Ehrlichia spp. morulae, and to compare patterns of lymphoid reactivity between dogs with CME and those with noninfectious causes of lymphoid hyperplasia. Methods: Cytologic preparations of LNs from 35 dogs with nonmyelosuppressive CME (group A), 16 dogs with myelosuppressive CME (group B), 26 dogs with noninfectious diseases (group C), and 15 healthy dogs (group D) were evaluated. Percentages of lymphocyte types, plasma cells, macrophages, neutrophils, and eosinophils were determined. Samples from dogs in groups A and B were evaluated for the presence of morulae. Results: Cytologic abnormalities in LNs were recorded in 54% of dogs in group A, 88% in group B, 39% in group C, and 0% in group D and were more frequent (P=.02) in dogs with myelosuppressive CME than those with nonmyelosuppressive CME. Plasma cell hyperplasia was more frequent in CME than in noninfectious diseases (P=.03). An association between the presence of cytologic abnormalities and morulae in group A dogs was not found. Conclusions: Dogs with myelosuppressive CME have more lymphoid cytologic abnormalities than dogs with nonmyelosuppressive CME. LN plasmacytosis is the major pattern of lymphadenopathy in dogs with CME and is found more frequently in dogs with CME than in dogs with noninfectious causes of lymphadenopathy.