Canine vector-borne disease pathogens in dogs from south-east Queensland and north-east Northern Territory

Objectives To determine the prevalence of canine vector‐borne diseases (CVBD: Babesia spp., Anaplasma spp., Ehrlichia spp., haemotropic mycoplasmas and Hepatozoon) in Australian dogs; namely, dogs from pounds in south‐east Queensland and an indigenous Aboriginal community in the north‐east of the Northern Territory. Design and procedure Blood samples were collected from 100 pound dogs and 130 Aboriginal community dogs and screened for the CVBD pathogens using polymerase chain reaction (PCR). All positive PCR products were sequenced for species confirmation. Results In total, 3 pound dogs and 64 Aboriginal community dogs were infected with at least one CVBD pathogen. Overall, B. vogeli was detected in 13 dogs, A. platys in 49, M. haemocanis in 23, Candidatus Mycoplasma haematoparvum in 3 and C. M. haemobos in 1 dog. Co‐infections were detected in 22 Aboriginal community dogs. Conclusions This study found B. vogeli, A. platys and haemotropic mycoplasma infections to be common in dogs in subtropical and tropical areas of Australia. This study also reports for the first time the prevalence and genetic characterisation of haemotropic mycoplasmas in dogs in Australia.     

Detection of Ehrlichia canis by polymerase chain reaction in dogs from Portugal

Antibodies against Ehrlichia canis, the cause of canine monocytic ehrlichiosis, have been reported previously in clinically ill and stray dogs from Portugal. In this study, the 16S rRNA gene of E. canis was detected by the polymerase chain reaction (PCR) in 12/55 (22%) of dogs with suspected tick-borne disease in the Algarve region in Portugal.     

A newly developed droplet digital PCR for Ehrlichia canis detection: comparisons to conventional PCR and blood smear techniques

Canine monocytic ehrlichiosis caused by Ehrlichia canis infection is a life-threatening vector-borne disease in dogs worldwide. Routine blood smear has very low sensitivity and cannot accurately provide a quantitative result. Conventional PCR (cPCR) and real-time PCR (qPCR) are widely used as molecular methods for E. canis detection. qPCR is quantitative but relies on standard curves of known samples. To overcome this difficulty, this study developed a new E. canis quantitative detection method, using droplet digital polymerase chain reaction (ddPCR). ddPCR was evaluated against cPCR and blood smears. PCR amplicons and genomic DNA (gDNA) from 12 microscopic positive samples were used to identify the limits of detection (LODs) in ddPCR and cPCR. Our ddPCR was assessed in 92 field samples, it was compared with cPCR and blood smears. ddPCR showed LOD=1.6 copies/reaction, or 78 times more sensitive than cPCR (LOD=126 copies/reaction), using PCR amplicons as a template, whereas both ddPCR and cPCR had equal LODs at 0.02 ng gDNA/reaction. In addition, ddPCR had 100% sensitivity and 75% specificity for E. canis detection compared to cPCR and no cross-reaction with other blood pathogens was observed. ddPCR identified more positive samples than cPCR and blood smear. ddPCR improved the overall performance of E. canis detection, with a better LOD and comparable sensitivity and specificity to cPCR. The technique might be helpful for diagnosis of E. canis in light infection, evaluating the number of E. canis and follow-up after treatment.     

Anemia of inflammation in dogs infected with Ehrlichia platys

Ten adult male dogs were inoculated with Ehrlichia platys, and blood samples were collected throughout the infection to evaluate the hematologic changes with respect to serum biochemical analytes. All dogs developed a mild, normocytic, normochromic anemia by postinoculation day 7, with significantly (P less than 0.05) decreased serum iron concentration and total iron-binding capacity. Stainable bone marrow iron appeared normal or increased throughout the infection. By postinoculation day 31, the PCV was not significantly different from the pretreatment value. All dogs became hypergammaglobulinemic, leukopenic, hypoalbuminemic, and hypocalcemic during the infection. These findings were compatible with the syndrome of anemia of inflammation.     

Serological survey for Ehrlichia canis in urban dogs from the major population centres of northern Australia

OBJECTIVE: To detect evidence of Ehrlichia canis infection of dogs from the major population centres of northern Australia, if present. DESIGN: Serological investigation for E. canis. PROCEDURE: The sera of 316 domestic dogs, collected from the northern Australian population centres of Townsville, Cairns, Darwin, Kununurra and Broome from May 1997 to August 1999, were investigated for evidence of infection with E. canis. Samples were tested for antibodies to E. canis using an indirect fluorescent antibody (IFA) test. The buffy coats from blood of dogs whose serum reacted in the IFA test were subsequently tested with a nested PCR to detect E. canis DNA. When available, blood from these dogs was injected into suckling mice, which were then examined for clinical disease and tested for the presence of E. canis antibodies. RESULTS: Of the 316 samples tested seven reacted in the IFA test for E. canis. None of the dogs from which these samples were obtained exhibited clinical signs of acute or chronic ehrlichiosis. The six positive samples available for testing were negative when tested with the nested PCR. Suckling mice inoculated with blood from three of the dogs whose serum was positive by IFA test showed no signs of clinical disease nor did their give positive reactions in the IFA test. CONCLUSIONS: No evidence of E. canis infection was confirmed in any of the dogs examined. Northern Australia would appear to remain free of this obligate parasite.     

Seroprevalence of Ehrlichia canis infections in police dogs in Spain

A seroepidemiological study of Ehrlichia canis was made in police dogs in Madrid (Spain). AntiEhrlichia canis antibodies were detected by indirect fluorescent antibody test for the detection of IgG.

The results obtained (three positive dogs out of a population of 131 animals) represents a seroprevalence of canine ehrlichiosis of 2.29%. This seroprevalence is one of the lowest described for this type of population.

The seroprevalence obtained from hunting dogs kennelled in Madrid in 1993 was 66.7% (24 positive out of a population of 36). Environmental conditions (temperature, humidity, rainfall, etc.) in both populations were very similar. We suggest that these conflicting results are due to the different prophylactic programmes used in these two populations.     

利用16S rDNA快速鉴定C群链球菌的研究

本试验应用PCR方法,根据16S rDNA两端的保守序列,用primer5.0软件设计引物扩增待检细菌,然后对扩增产物DNA片段进行测序、序列分析,经与Gen Bank中的已知序列进行同源性比较后,发现该未知菌属于链球菌C群。该方法比传统的细菌鉴定方法省时省力,并且能将细菌定型到群,结果准确、快速、重复性好。     

大肠杆菌属16S rDNA实时荧光定量PCR方法的建立及应用

根据GenBank大肠杆菌属16S rDNA基因序列设计并合成PCR引物及针对大肠杆菌属的特异Taqman探针,以大肠杆菌标准菌株的PCR扩增产物作为阳性模板制定标准曲线,建立大肠杆菌属实时荧光定量PCR检测方法。该法Mg2＋最佳工作浓度5 mmol/L,探针最佳工作浓度0.12μmol/L,标准曲线相关系数为0.998,能够定量和特异性检测大肠杆菌而与肠道优势菌群的代表种葡萄球菌、双歧杆菌和芽孢杆菌呈阴性反应,检测大肠杆菌16S rDNA基因的灵敏度达40.8 copies/μL。应用建立的方法对20、26、324、1、47、56日龄樱桃谷鸭的气管、食道、十二指肠、空肠、回肠、盲肠和直肠进行检测结果表明（以每个细菌含1个16S rDNA基因拷贝数计）,大肠杆菌属细菌总量,气管内为7.06×10^7～3.01×10^8个;食道内为1.73×10^8～3.23×10^8个;十二指肠内为2.29×10^8～2.39×10^9个;空肠内为1.28×10^8～1.69×10^9个;回肠内为1.86×10^8～1.90×10^10个;盲肠内为6.01×10^8～1.38×10^9个;直肠内为1.07×10^8～4.67×10^10个。樱桃谷鸭从食道到直肠的大肠杆菌属细菌的数量呈现上升趋势,盲肠和直肠分布量较多。     

Ruminal distribution of the cellulolytic bacterium Fibrobacter succinogenes in relation to its phylogenetic grouping

To investigate the ecological importance of the cellulolytic bacterium Fibrobacter succinogenes in fiber digestion, ruminal distribution of F. succinogenes was determined in relation to its phylogenetic grouping. Rumen digesta from wethers and steers fed orchardgrass hay, rice straw or fresh orchardgrass were employed as the materials for the analyses. Orchardgrass hay stem incubated in the rumen was also used. By using total DNA extracted from these materials, population sizes of total F. succinogenes and of four different phylogenetic groups of this species were quantitated through competitive polymerase chain reaction (PCR), and restriction fragment length polymorphism analysis of PCR products targeted the bacterial 16S rDNA. Rumen digesta and ruminally incubated hay stems had a reasonably high population size of F. succinogenes (×10^7?8/g) that was composed of strains belonging to the phylogenetic groups 1 and 3. The relative abundance of each group was different among the samples; group 1 dominated on the ruminally incubated hay stem and in the rumen of wethers fed fresh orchardgrass, while group 3 was major in the rumen of wethers and steers on hay diet. These results suggest that there could be phenotypic differences among the phylogenetic groups of F. succinogenes, and group 1 dominating on hay stem might contribute to rumen fiber digestion more than the other groups.     

Experimental transmission of granulocytic ehrlichial organisms in dogs

Canine granulocytic ehrlichial organisms were transmitted from an infected dog from Missouri to two male, 10-month-old dogs by an intravenous injection of whole blood. Physical or behavioral abnormalities were not detected during the 98 days of evaluation other than a mild pyrexia from Day 18 to 20. Ehrlichial morulae were found in blood granulocytes of Dog 1 from Day 13 to 44 and of Dog 2 from Day 14 to 34 with the peak rickettsemia occurring on Day 16 for both dogs. By Day 21 after inoculaiuon, both dogs had positive titers to Ehrlichia canis. The highest titers for both dogs were found 63 days after inoculation, after which the titers decreased. Most of the hematologic abnormalities (i.e., neutropenia, lymphocytosis, thrombocytopenia) and fever occurred between 18 and 24 days after inoculation. The pathologic bases of these abnormalities were not investigated but their concurrent presence suggested an association with the dogs' immunologic responses to the granulocytic ehrlichial agent. Results from the study indicated that the canine granulocytic ehrlichial agent of Missouri may produce subclinical infections and suggested that dogs may be able to clear the organism without antimicrobial therapy.     

Monitoring C-reactive Protein in Beagle Dogs Experimentally Inoculated with Ehrlichia canis

The concentrations of C-reactive proteins (CRP) in the plasma of five beagle dogs experimentally inoculated with Ehrlichia canis increased markedly. The concentrations began to increase between 4 and 16 days and peaked between 15 and 42 days after inoculation of E. canis. The peak concentrations ranged from 217.8 to 788.8 g/ml (452.6±228.1 SD). After the peak, the concentrations of CRP decreased rapidly. The PCR product of 16S rRNA of E. canis became detectable in the five dogs between 18 and 27 days after inoculation of E. canis. Antibodies to E. canis were detected in plasma from the dogs between 5 and 15 days after inoculation of E. canis. The timings of seroconversion and of the start of the increase in CRP were approximately similar and the high concentrations of CRP in the plasma of the dogs tended to become apparent when the PCR product of 16 S rRNA of E. canis became detectable.     

Genetic characterization of Anaplasma (Ehrlichia) platys in dogs in Spain

This paper reports the first genetic characterization of Anaplasma (Ehrlichia) platys in Spain from a naturally infected dog. The dog presented clinical signs compatible with canine ehrlichiosis. After DNA extraction and PCR amplification, 16S rRNA gene and citrate synthase gene ( gltA) of this agent were amplified. The GenBank accession number for the nucleotide sequence of the 16S rRNA gene of this strain is AY530806. The A. platys strains registered in France and Japan showed the highest similarity to the 16S rRNA gene sequence obtained from the Spanish strain. In the amplification of the gltA gene, a 1443 bp fragment was obtained, and three nucleotide differences were detected in comparison with other strains sequences. These data confirm the presence of A. platys in a dog showing clinical signs compatible with ehrlichiosis in Spain.     

Doxycycline clearance of experimentally induced chronic Ehrlichia canis infection in dogs

BACKGROUND: Ineffective clearance of Ehrlichia canis after doxycycline administration has been reported despite the fact that the recommended treatment for canine ehrlichiosis is doxycycline. The effectiveness of doxycycline in clearing E canis infection from the blood and tissues of dogs requires additional evaluation. HYPOTHESIS: Doxycycline (5 mg/kg PO q12h), administered for 4 weeks, will eliminate E canis infection from the blood and tissues of experimentally infected dogs. ANIMALS: Fifteen Walker hound-mixed breed dogs were inoculated subcutaneously with E canis-infected canine histiocytic cells 4 months before doxycycline treatment. METHODS: Four dogs were treated with doxycycline (5 mg/kg PO q12h for 3 weeks), 5 dogs were treated with doxycycline at the same dosage for 4 weeks, and 5 control dogs were not treated. Dexamethasone (0.4 mg/kg i.v.) was given after treatment to precipitate recrudescence of any remaining E canis organisms. Platelet counts, anti-E canis immunofluorescent antibodies, and polymerase chain reaction (PCR) detection of E canis deoxyribonucleic acid (DNA) in blood and tissues were evaluated. RESULTS: E canis DNA was not detected in the blood and tissues of doxycycline-treated dogs after treatment. Platelet counts were within reference intervals, and E canis antibodies decreased. Spontaneous clearance of E canis infection occurred in 2 of 5 control dogs. Three control dogs had E canis DNA detected in blood and tissues, platelet counts remained low or within the reference interval, and E canis antibodies remained high. CONCLUSIONS AND CLINICAL IMPORTANCE: As administered in this study, doxycycline cleared E canis from the blood and tissues of experimentally infected dogs.     

Prevalence of Ehrlichia canis infection in thrombocytopenic dogs from Rio de Janeiro, Brazil

BACKGROUND: Infection with Ehrlichia canis causes a highly variable, multisystemic disease in dogs. Nevertheless, many clinicians in Rio de Janeiro, Brazil, use the presence of only thrombocytopenia to make a presumptive diagnosis of E canis infection. OBJECTIVE: The objective of this study was to determine the prevalence of E canis in thrombocytopenic dogs from Rio de Janeiro, Brazil, using polymerase chain reaction (PCR). METHODS: Following DNA extraction of whole blood samples from 226 dogs, PCR assays were done using primers for rickettsial DNA (including Ehrlichia spp, Anaplasma platys and A phagocytophilum) and using E canis-specific primers (16S rRNA gene). Dogs were grouped as thrombocytopenic and nonthrombocytopenic based on platelet counts. The null hypothesis that there was no difference in the prevalence of E canis in these groups was rejected at P<.05. RESULTS: Thirty-six (32.1%) of the thrombocytopenic dogs and 4 (3.5%) of the nonthrombocytopenic dogs were positive for rickettsial gene sequences (P<.0001). Further, 30 (26.8%) of thrombocytopenic dogs and 4 (3.5%) nonthrombocytopenic dogs were positive for E canis-specific gene sequences (P<.0001). CONCLUSIONS: Although the prevalence of E canis infection was higher in thrombocytopenic dogs, less than one third of these dogs had demonstrable E canis infection. Thus, thrombocytopenia is not specific for the detection of E canis infection and should not be used solely to establish a diagnosis of canine ehrlichiosis, even in a geographic area with relatively high disease prevalence.     

Serological investigation of granulocytic Ehrlichia infection in sheep in Norway

Serum samples of 749 sheep from 75 sheep flocks in Norway, i.e. 361 lambs (6 to 7 months old) and 388 adults (>1.5 year), were analysed for antibodies to Ehrlichia equi. Ten animals from each flock were examined. Seropositive animals were found along the coast of southern Norway from Vestfold to Sør-Trøndelag (as far north as 63°38''N). Seropositive sheep were not found in southeast, east or northern Norway. Thirty-two flocks were seropositive, although tick-borne fever had only been diagnosed earlier in half of these. In 78% of the seropositive flocks, more than 80% of the sheep were seropositive. A total of 35.7 % and 36.3 % of lambs and adults were found seropositive, respectively. However, the overall seroprevalence among animals that had been grazing on Ixodes pastures were 0.80 for the lambs and 0.84 for the adults. Mean antibody titres (± SD) (log₁₀) in seropositive lambs and adults were 2.59 (± 0.449) and 2.70 (± 0.481), respectively. No significant differences in either seroprevalence or mean antibody titre between sheep of different ages were obtained in this study. Based on antibodies 94% of sheep flocks on Ixodes pastures were infected with a granulocytic Ehrlichia infection. The association between seropositive flocks and Ixodes infested pasture shows a very high degree of agreement (p < 0.00001). The present study indicates that granulocytic Ehrlichia infection in sheep is underdiagnosed in Norway.     

吉林白鹅消化道纤维分解菌的鉴定

选择54日龄吉林白鹅(肉用),无菌刮取盲肠内壁黏膜,筛选菌株进行分离培养,应用DNS法测定酶活力,通过菌株生化试验及16S rDNA测序同源性比较,鉴定出具有产纤维素酶特性的菌株,利用PCR进行筛选,基因组部分测序,结果经序列BLAST比对,确定其部分序列与无芽孢杆菌同源性达到99%,初步鉴定为无芽孢杆菌属。     

斑点叉尾(鮰)嗜麦芽寡养单胞菌的分离鉴定及系统发育分析

从发生在四川、重庆等省市的斑点叉尾(鮰)急性流行性传染病病鱼的肝脏、肾脏内分离到1株高致病性菌株(CCF00024),人工感染健康鱼表现出与自然病鱼相同的症状,并从中分离获得同种细菌,证实其为斑点叉尾(鮰)急性流行性传染病的病原菌.形态、生理生化检测表明,该菌为非发酵型直杆菌,严格需氧,革兰氏阴性,极生多鞭毛,对除麦芽糖和甘露糖以外的多种糖类不利用产酸,氧化酶阴性,DNA酶、蛋白酶、脲酶、赖氨酸脱羧酶阳性,MR、VP阴性.在以该菌16S rDNA序列(GenBank登录号AY970826)和GenBank及RDP数据库内同源性较高的细菌16S rDNA序列构建的系统发育树中,分离菌CCF00024与嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia)聚在一簇,特别是与S.maltophilia M5-1的同源性最高,序列相似性达99.6%,结合形态和生理生化特点将其鉴定为嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia).药敏试验结果表明,对磺胺甲(口恶)唑、磺胺异(口恶)唑、阿齐霉素、洛美沙星高度敏感,而对新霉素、卡那霉素、氨苄青霉素,头孢唑啉、先锋霉素V和链霉素不敏感.