Cutaneous sterile pyogranuloma/granuloma syndrome in a dog

A case of canine cutaneous sterile pyogranuloma/granuloma syndrome (SPGS) with generalized asymmetrical alopecia and plaques is described. Results from histopathologic examination were suggestive of SPGS and failed to demonstrate any etiological agent. A systematic approach on how to definitively rule out infectious skin diseases is discussed.     

Diagnosis of canine Hepatozoon spp. infection by quantitative PCR

Hepatozoon (H.) americanum and H. canis are the etiological agents of canine hepatozoonosis, a disease that is found worldwide and is also prevalent in the southeastern United States. Current laboratory diagnosis of canine hepatozoonosis caused by H. americanum is usually dependent on visual identification of Hepatozoon "onion skin cysts" in muscle biopsies, an approach that requires invasive sampling and can result in false negatives. We have developed a diagnostic method for detection of Hepatozoon spp. DNA that integrates nucleic acid extraction with extensive agitation to maximize DNA extraction efficiency. The DNA extracted from canine EDTA-whole blood is subjected to real-time PCR, and fluorescence resonance energy transfer (FRET) probes detect a signature polymorphism in the amplified DNA. This PCR method amplifies a fragment of the Hepatozoon 18S rDNA gene, detects as few as 7 genomic copies of Hepatozoon spp. per ml of blood with high specificity, and differentiates between H. americanum and H. canis amplicons. A surprising 300-fold increase of H. americanum 18S rDNA targets occurred during 3-0 days of storage of positive blood specimens. Examination of 614 EDTA-blood samples submitted mostly from the southeastern Unites States from dogs with suspected hepatozoonosis identified H. americanum in 167 samples (27.2%). An additional 14 samples (2.3%) were positive for H. canis, and 14 samples (2.3%) were positive for both H. americanum and H. canis. These results suggest that the Hepatozoon spp. 18S rDNA quantitative PCR may be a valuable tool that can improve diagnosis and therapy of canine hepatozoonosis.     

Risk of infection with Leishmania spp. in the canine population in the Netherlands

The dog is the main reservoir of Leishmania infantum, the causative agent of visceral leishmaniasis (VL) in humans in Southern Europe. In order to identify the risk of dogs from a Leishmania non-endemic area traveling to a Leishmania-endemic area becoming infected and the risk of transmitting infection to humans in non-endemic areas an investigation was performed, in which the results of a questionnaire were combined with the results of a serologic survey. The questionnaire was sent to 1478 at random chosen families in the Netherlands. Of the 59.0% responders 28.0% had one or more dogs and 4.8% of these dogs had visited Southern Europe during the summer period of that year. On a total population of 1,200,000 dogs in the Netherlands, this means that each year some 58,000 dogs are at risk of being exposed to a Leishmania infection in Southern Europe. During the period 1990-1992 blood was collected for serology in 1911 dogs presented to the Utrecht University Clinic because of clinical problems not related to leishmaniasis, of which 434 had been in Southern Europe in the foregoing years. None was serologically positive. From these data it can be deduced that the highest chance to obtain leishmaniasis during a vacation in Southern Europe is mathematically less than 1/434 or less than 0.23%. Serology was also performed during the period 1989-1993 in 597 dogs that had been in Southern Europe and were suspected of leishmaniasis. Titers were positive in 145 of these samples. Sixty-four of these dogs were born in the Mediterranean and had been imported into the Netherlands. Excluding these imported dogs, it was calculated that at least 0.027% of the 58,000 dogs yearly taken to Southern Europe during holidays become infected with Leishmania. In order to establish the risk of disease transmission for people in close contact with an infected dog, serum samples of owners and house mates of 37 dogs with leishmaniasis were tested. All 112 sera tested negative. It was concluded that the risk to get leishmaniasis was between 0.027% and 0.23% for the dog when taken to Southern Europe during vacation, and that the risk for owners in non-endemic areas to get leishmaniasis from an infected dog is minimal.     

Confirmation of canine leproid granuloma syndrome in New Zealand

Canine leproid granuloma syndrome (CLGS) has not been officially reported in New Zealand. The seminal report describing this syndrome is in the Australian Veterinary Journal, 1998, where the results of a questionnaire circulated amongst veterinary pathologists and practitioners in Australia were reported. It included one response of a case seen in New Zealand, but no details of that case were given, despite CLGS being described in the literature as “common in New Zealand”. By injudicious use of references, the international literature has propagated the idea that the condition, including molecular identification, was confirmed in New Zealand, yet none of the articles cited actually confirmed that. An outbreak of skin granulomas in a group of approximately 35 working dogs was investigated, in which skin samples were sent to the Mycobacterium reference laboratory, Victoria, Australia, for PCR testing and molecular characterisation. Results of the clinical presentation, histological features and molecular studies conformed to the published details of CLGS. In particular, the nucleotide sequence of the internal transcribed spacer region, amplified from the mycobacterial DNA present in the clinical specimen provided, was identical to GenBank® Accession Number EF611177. That sequence is representative of the causative agent of CLGS in cases from Australia, the United States of America and Brazil. Although acid-fast organisms are occasionally seen in skin granulomas in dogs in New Zealand, this is the first confirmed identification of CLGS in this country. This is also the first report of an outbreak situation amongst a group of dogs.     

PCR identification of Leishmania in diagnosis and control of canine Leishmaniasis

Leishmaniases are endemic in many countries, mainly in rural areas. In Brazil, Leishmania infection is responsible for many cases of Leishmaniases, including recent reports in urban regions. Despite their sensitivity, traditional serological and parasitological methods for detecting Leishmaniases have proven inadequate for species discrimination. This study aimed to identify Leishmania species in biological samples by a fast methodology, avoiding "in vitro" cultivation. Knowledge of the Leishmania species is an important tool in regions where both New World visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) are prevalent. As these new foci appear in areas not traditionally endemic for VL, the main problem is to distinguish between true autochthonous infections and infections acquired in other well-known endemic areas. Since, domestic dogs are known to be the main VL and CL reservoir, they are regularly investigated in endemic areas to prevent, principally, severe and often fatal VL in humans. However, several infected dogs present no clinical signs or clinical signs similar to other canine diseases. Here, we evaluated the ability of PCR to diagnose VL and distinguish L. (L.) chagasi from other Leishmania species in domestic dogs. Samples from 114 dogs from 30 cities (Sao Paulo, Brazil) were divided into two groups: 44 symptomatic and 70 asymptomatic. They were assayed by parasitological methods (culture and microscopic examination) and PCR to determine L. (L.) chagasi, L. (V.) braziliensis; and in some cases, Leishmania spp. Parasitological tests and PCR-L. chagasi were concordant in 105 samples (92%). VL was confirmed in 49 dogs, while 56 had negative results. Of the 114 samples, 9 had discordant results, but were further tested by PCR-Leishmania spp. with positive results. VL was also confirmed in 4 dogs having negative parasitological tests and positive PCR-L. chagasi. Consequently, this PCR was positive for 100% (53/49) of dogs with parasites detected in parasitological tests. Also, PCR demonstrated high specificity detecting 61 dogs negative for VL. Leishmania infection was negative in 56 dogs, and 5 with positive culture and PCR-Leishmania spp. had CL since they were positive in PCR-L. braziliensis. This study shows the importance of including PCR in diagnosis of Leishmaniases by differential diagnosis contributing to the surveillance and control of VL programs.     

Detection of Leishmania infantum by real-time PCR in a canine blood bank

O bjectives : Risk for transmission of Leishmania infantum from blood products has been largely demonstrated in human and veterinary literature. Appropriate screening of canine blood donors is important especially in an endemic area such as Barcelona (Spain). The purpose of this study was to evaluate the presence of L infantum DNA parasites by real-time quantitative PCR in our canine blood bank.
M ethods : Samples from blood products obtained from 92 canine blood donors were assayed for L infantum by means of real-time PCR amplification and quantification.
R esults : The prevalence of quantitative PCR-positive blood samples among healthy seronegative blood donors was 19·6 per cent.
C linical S ignificance : The results of this study show that L infantum infection is common in canine blood donors and their blood products in an endemic area, despite a negative commercial serological screening for infectious diseases. Therefore, screening by PCR should be included in an integrated approach to evaluate L infantum infection among potential blood donors.     

Differentiation between canine cutaneous and visceral leishmaniasis by the detection of immunoglobulin G specific for Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi antigens using flow cytometry

Flow cytometry employing Leishmania (L.) chagasi (Lc) and L. (Viannia) braziliensis (Lb) antigen was used to establish the differential diagnosis between visceral (VL) and cutaneous leishmaniasis (CL) in dogs. Flow cytometry permitted the detection of Leishmania-specific immunoglobulin G in sera from 19 dogs: nine with CL and 10 with VL. A significant difference in the percentage of positive staining was observed in sera from dogs with CL between the homologous antigen (69% for Lb) and the heterologous antigen (42% for Lc). However, this difference was not significant in sera from dogs with VL (61% for Lb and 73% for Lc). No significant staining was observed in control sera (0.6% for Lb and 0.4% for Lc) consisting of samples from healthy dogs, or in the group with sporotrichosis (1.8% for Lb and 1.5% for Lc), a differential diagnosis of CL. The results suggest that flow cytometry might be useful for the differentiation between CL and VL in dogs, with practical applications in areas where the two infections overlap.     

Treatment of canine leproid granuloma syndrome: preliminary findings in seven dogs

OBJECTIVE: To determine effective treatment strategies for patients with refractory canine leproid granuloma syndrome. DESIGN: Multi-institutional retrospective/prospective case series using client-owned dogs. PROCEDURE: Seven dogs (four Boxers, one Dobermann, one Bullmastiff and one Bullmastiff cross-bred; ages 3 to 11 years) with leproid granulomas were treated successfully using a variety of treatment regimens. These cases were recruited because: lesions were either widely distributed over the dog; progressive, despite routine therapy, or were associated with particularly disfiguring lesions. The treatment regimen evolved during the course of the clinical study. RESULTS: Combination therapy using rifampicin (5 to 15 mg/kg p.o., every 24 h) and clarithromycin (8 to 24 mg/kg p.o. daily; dose divided every 8 or every 12 h) was used most frequently and proved to be effective and free from side effects. Total daily doses of clarithromycin in excess of 14 mg/kg were considered optimal and long treatment courses, in the order of 1 to 3 months, were used. Combination therapy using rifampicin (25 mg/kg; that is, higher than the recommended dose) and clofazimine was effective in one case, but resulted in hepatotoxicity. A topical formulation of clofazimine in petroleum jelly was used as an adjunct to oral rifampicin and doxycycline in another patient treated successfully. CONCLUSION: Based on our evolving clinical experience, a combination of rifampicin (10 to 15 mg/kg p.o., every 24 h) and clarithromycin (15 to 25 mg/kg p.o. total daily dose; given divided every 8 to 12 h) is currently recommended for treating severe or refractory cases of canine leproid granuloma syndrome. Treatment should be continued (typically for 4 to 8 weeks) until lesions are substantially reduced in size and ideally until lesions have resolved completely. A topical formulation, containing clofazimine in petroleum jelly may be used as an adjunct to systemic drug therapy. Further work is required to determine the most cost effective treatment regimen for this condition.     

PCR for detection of Mycobacterium tuberculosis in experimentally infected dogs

The susceptibility of dogs to infection with Mycobacterium tuberculosis was studied through different ways of experimental infection. The examination shows, that in most cases the disease runs subclinically with pathological changes localized mainly in the lungs, lymph nodes, small intestines, liver, kidneys and spleen. Histological findings demonstrate granulomatous inflammation with caseosation and predominance of epitheloide macrophages and single lymphocytes. Tissue samples from internal organs of experimentally infected dogs as well as non-infected but contact animals were investigated by direct PCR. Specific PCR-products were obtained in 44 of 96 studied samples. Eighty-three (86.5%) of PCR results coincided with bacteriological finds, 82 (85.4%) with the pathological and 71 (74.0%) simultaneously with bacteriological and pathological results. The observed specific DNA products in tissue samples of infected and non-infected dogs demonstrate significant sensitivity of PCR method. It could be assumed that the transmission of M. tuberculosis infection is possible by close contact between ill and healthy dogs and that the naturally infected dogs or dogs suffering from tuberculosis may serve as a permanent source of infection to humans and other animals.     

A multiplex PCR for the detection of Brucella spp. and Leptospira spp. DNA from aborted bovine fetuses

Bovine brucellosis and leptospirosis are important causes of bovine abortion around the world. Both diseases can be serologically diagnosed, but many factors may cause false positive and negative results. Direct methods based on bacteriological isolation are usually employed, but they are difficult, time consuming and dangerous. Monoplex polymerase chain reaction (PCR) have been successfully described for the detection of Brucella spp. and Leptospira spp. Aiming at improvement in the direct diagnosis, a multiplex PCR (mPCR) for the detection of these agents in aborted bovine fetuses is described. The detection threshold of the mPCR was evaluated in experimentally contaminated bovine clinical samples using a conventional proteinase K/SDS or a boiling-based extraction protocols. The mPCR was applied to two groups of clinical samples: 63 episodes of bovine abortion and eight hamsters experimentally infected with Leptospira interrogans serovar pomona. Adopting microbiological isolation as reference, the test showed a sensitivity of 100% in both groups of clinical samples. Seven samples collected from bovine fetuses were Brucella spp. culture negative but showed positive results in mPCR. Regarding Leptospira spp. detection, similar results were observed in three bovine clinical samples. All hamsters infected with Leptospira were positive in both microbiological culture and mPCR. The boiling extraction protocol showed better results in some clinical samples, probably by the removal of PCR inhibitors by heat treatment. The high sensitivity, simplicity and the possibility of detection of both bacteria in a single tube reaction support the use of the mPCR described in the routine diagnosis.     

Evaluation of the presence of Leishmania spp. by real-time PCR in the lacrimal glands of dogs with leishmaniosis

Leishmania infantum infection is highly prevalent in endemic areas. Dogs with leishmaniosis may develop keratoconjunctivitis sicca (KCS). The goals of this study were (1) to quantify Leishmania amastigotes in the Meibomian glands (MG), main lacrimal gland (MLG) and nictitating membrane gland (NMG) from dogs with leishmaniosis; (2) to compare these results to immunohistochemistry (IHC), and (3) to explore the association between the Leishmania parasite load and the presence of ocular clinical signs. Twenty-five dogs diagnosed with leishmaniosis were included. MG, MLG and NMG from both eyes were collected. Histopathology, IHC and real-time PCR were performed. All specimens yielded positive real-time PCR results. For all three glands, samples from dogs with ocular clinical signs had mean ΔCt (cycle threshold) values significantly lower (higher parasite loads) than those from dogs without signs. Cut-off values of ΔCt<0, ΔCt<4 and ΔCt<4.9 for MG, MLG and NMG, resulted in a likelihood ratio of positives of 5.9, 6.38 and 6.38, respectively. Samples with ΔCt values below the reported cut-off were significantly more likely to display clinical signs related to KCS than those with results above the cut-off, for all three glands. Similarly, ΔCt values below the cut-off were significantly associated with positive IHC. In this study real-time PCR has been standardised for use in MG, MLG and NMG. A cut-off value established for each of these tissues may aid the clinician in the discrimination between ocular signs related to Leishmania from those associated with other causes of KCS.     

Moprhological diversity of cultured canine gastric Helicobacter spp.

The cell morphology, the number of flagella, the occurrence of periplasmic fibrils and ultrastructural structures of five groups of cultured canine gastric Helicobacter spp. were compared. The study included four strains of Helicobacter felis, four strains of Helicobacter bizzozeronii, one strain of ‘Flexispira’, six strains of an unnamed spiral organism 2 and one strain of an unnamed spiral orgaism 3 which were isolated from gastric biopsies. Cultures were studied with negative staining, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Bacterial dimensions were measured from the negative staining samples and values were tested with ANOVA and Bonferroni tests. The organisms studied differed from each other morphologicall. H. felis was a slightly spiraled organism with periplasmic fibrils. ‘Flexispira’ was a thin and straight organism with periplasmic fibrils. H. bizzozeronii was a tightly spiraled organism. Spiral organism 2 was loosely spiraled and thicker than the other organisms. Spiral organism 3 was a short curved rod having a single bipolar flagellum. The other species had multiple flagella. As a conclusion the canine gastric Helicobacter spp. can be differentiated from each other morphologically with an electron microscope. The morphological differences were mainly found in the structures involved in motility. The importance of the differences may lie in their impact on the colonization in a gastric mucous environment.     

Improved detection of Mycobacterium avium subsp. paratuberculosis In milk by immunomagnetic PCR

The potential use of a novel immunomagnetic PCR (IMS-PCR) technique as a rapid method to screen milk samples for the presence of Mycobacterium avium subsp. paratuberculosis (M. ptb) was assessed. Immunomagnetic separation (IMS) for M. ptb, developed at Queen's University, Belfast, was applied to milk samples prior to IS900 PCR in order to selectively concentrate any M. ptb cells present and, at the same time, separate the cells from constituents of milk likely to inhibit subsequent PCR. This increased the sensitivity of IS900 PCR. IMS-PCR sensitivity could be further increased by initial centrifugation (2500 g for 20 min) of larger volumes of milk (10 and 50 ml), and resuspension of the sediment into a 1 ml volume appropriate for IMS treatment. Following IMS, template DNA for IS900 PCR was obtained by heating the bead-cell suspension in a thermal cycler at 100 degrees C for 15 min. It was estimated that the IMS-PCR assay could detect approximately 10(3)CFU of M. ptb per 50 ml of milk (equivalent to 20 CFU/ml), whereas the minimum detection limit of direct IS900 PCR was estimated at 10(5)CFU of M. ptb per 50 ml (equivalent to 2000 CFU/ml). A blind trial was carried out in which a total of 40 spiked (10(6)CFU M. ptb) and unspiked, raw and laboratory-pasteurised milk samples were independently tested by IMS-PCR and conventional IS900 PCR. IMS-PCR correctly identified 97. 5% of milk samples (sensitivity 100%, specificity 95%), including spiked milk samples before and after laboratory-pasteurisation. One false positive result was obtained which may have resulted from carryover between samples during the IMS procedure. Conventional IS900 PCR correctly identified only 72.5% of the same 40 milk samples (sensitivity 23%, specificity 100%). IMS-PCR was also shown to be capable of detecting natural M. ptb infection in raw sheep's milk, and raw and commercially pasteurised cows' milk.     

The detection and determination of potential virulence of Salmonella spp. using PCR method

The aim of this study was to prove that PCR is a very useful method to identify Salmonella strains and to determine their virulence factors by amplification of characteristic genetic markers. Investigations included 5 strains of Salmonella which were obtained from pure cultures and 1 Salmonella strain from the mixed culture. Genotypic analysis of 6 examined strains revealed the 163-bp fragment of chromosomal DNA, which is the DNA rep. ori. gene, encoding the particular genus. In all of these strains 215-bp, 203-bp and 204-bp chromosomal DNA fragments were identified as representing the stn, stpA and spaO genes that confirmed their virulence. These amplification products were identified in both pure and mixed culture from pork. Sensitive and rapid PCR method may be used not only for identification of Salmonella strains and for determination of their virulence factors but also for routine microbiological diagnostic of food pathogens.     

牛分枝杆菌特异性多重PCR反应在临床样本检测中的应用

本试验应用建立的三重PCR反应检测256份血液样本,结果其中的2份样本呈阳性.应用在线blast进行同源性比较,结果发现,牛分枝杆菌特异性基因IS6110,IS1081和recA与牛分枝杆菌AF2122/97株的同源性均达到99％以上.     

Diagnosis of Helicobacter spp. infection in canine stomach

A total of 75 biopsied samples of cardia, fundus, body, and pyloric antrum from necropsied dogs that were submitted to the Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University from April 2003 to June 2004 were investigated. The objectives of this study were to determine the prevalence of Helicobacter spp. in canine stomach by polymerase chain reaction (PCR) in comparison to histochemistry versus immunohistochemistry (IHC), and to correlate these diagnostic methods with the clinical significance in infected dogs. Histopathological results revealed 60.0% (45/75) of samples to be positive, and consisted of mild gastritis in 64.44% (29/45), moderate gastritis in 11.11% (5/45), and severe gastritis in 24.44% (11/45). The proportion showing no histopathological lesions was 40.0% (30/75). Helicobacter spp. were localized to the luminal crypt in 18.67% (14/75), gastric pit in 22.67% (17/75), gastric gland in 21.33% (16/75), and gastric epithelium in 8% (6/75). The percentages of positive samples of Helicobacter spp. diagnosed by hematoxylin and eosin stain (H&E), Warthin Starry stain (WSS), IHC with rabbit polyclonal anti-H. pylori antibody, and PCR were 17.3% (13/75), 46.7% (35/75), 30.7% (23/75), and 10.7% (8/75), respectively. No significant differences weree observed in histopathological changes in portions of the stomach (p>0.05). The diagnosis of Helicobacter spp. by PCR in comparison to that by WSS and IHC was not significantly different (p>0.05). There were no relationships between pathological studies using H&E, WSS, and IHC, and especially between PCR and clinical signs of Helicobacter spp. infections in canine stomachs (p>0.05). The present study revealed significantly different levels of correlation for Helicobacter spp. detection between H&E and WSS (p<0.001). Results indicate that the method of choice for diagnosis of Helicobacter spp. infection in canine stomach is dependent on the purpose of study and appropriate specimen collection.