Utility of diagnostic tests used in diagnosis of infection in dogs experimentally inoculated with a North American isolate of Leishmania infantum infantum

Eight female beagles were infected with 1 x 10(7) (low dose, LD) or 2 x 10(8) (high dose, HD) promastigotes of a North American isolate of Leishmania infantum infantum (LIVT-1 strain) isolated from naturally infected Virginia Foxhounds. Two female beagles served as negative controls and 2 male beagles chronically infected (> 3 years) with Leishmania infantum chagasi were positive controls. Bone marrow (BM) and lymph node (LN) aspirates were collected every 6-8 weeks for cytologic evaluation, parasite culture, and polymerase chain reaction (PCR). Serum samples were collected monthly for determination of serologic responses by indirect fluorescent antibody test (IFAT) and diagnostic rK39 antigen. Cultures of BM and LN aspirates and cytology evaluation were consistently positive in positive control dogs during the course of study. Negative control dogs were negative on BM and LN cultures and on cytologic evaluation of aspirates. Amastigotes were present on cytological examination of BM aspirates in 2 experimentally infected dogs. Cultures of LN aspirates were positive on 22 samples, whereas BM cultures were positive on 12 samples for all dogs. IFA titers ranged from 0 to 1 :400 in experimentally infected dogs during the course of the study. Recombinant K39 immunoassay tests were consistently positive in positive control dogs and in the HD dogs by approximately 8 weeks after infection. BM PCR products were identified more consistently in the HD dogs compared with the LD dogs. Kappa statistics indicated PCR correlated better with cultures and cytology than did IFAT or the rK39 immunoassay results in the experimentally infected dogs.     

Serologic and molecular evidence of coinfection with multiple vector-borne pathogens in dogs from Thailand

Forty-nine dogs from Thailand were evaluated for serologic evidence of exposure or polymerase chain reaction (PCR) evidence of infection with vectorborne pathogens, including Ehrlichia sp. (Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia equi, and Ehrlichia risticii), Bartonella vinsonii subsp. berkhoffi (Bvb), spotted fever group (SFG) rickettsiae (Rickettsia rickettsii), Typhus group (TG) rickettsiae (Rickettsia canada, Rickettsia prowazekii, and Rickettsia typhi), and Babesia sp. (Babesia canis and Babesia gibsonii). All study dogs had at least 1 of 3 entry criteria: fever, anemia, or thrombocytopenia. By immunofluorescence antibody (IFA) testing, seroreactivity was most prevalent to E chaffeensis (74%) and E canis (71%) antigens, followed by E equi (58%), Bvb (38%), E risticii (38%), R prowazekii (24%), B canis (20%), R rickettsii (12%), R canada (4%), and B gibsonii (4%) antigens. There was 100% concordance between E canis IFA and Western blot immunoassay (WI) for 35 of 35 samples; 2 samples were IFA and WI reactive only to E equi antigens. By PCR amplification, 10 dogs were found to be infected with E canis, 5 with Ehrlichia platys, and 3 with B canis. Sequencing of PCR products was undertaken to compare Ehrlichia strains from Thailand to strains originating from the United States. Partial DNA sequence analysis confirmed infection with E canis and E platys, with identical 16S rRNA sequence alignment to E canis (U26740) and to E platys (M83801), as reported in GenBank. Partial E canis P28.1 and P28.2 amino acid sequences from Thai dogs were divergent from analogous sequences derived from North American E canis (AF082744) strains, suggesting that the Thai dogs were infected with a geographically distinct strain of E canis compared to North American strains. The results of this study indicate that dogs in Thailand have substantial exposure to vectorborne diseases and that coinfection with these pathogens may be common.     

PCR法对伪狂犬病病猪不同部位的检测及敏感性试验

应用ＰＣＲ方法对ＰＲＶ感染细胞及自然发病猪不同组织样品进行检测,感染细胞毒最低检出量为１０５个ＴＣＩＤ５０病料组织样品最低取样为０１ｍｇ时,仍能得到阳性结果其敏感性显著高于微量血清中和试验。ＰＲＶ在自然发病猪体内分布很广,脑、三叉神经节（三叉Ｎ节）、嗅球、扁桃体、心脏、肝、脾、肺、肾均有ＰＲＶ的存在,ＰＲＶ检出率最高的组织为三叉神经节,其检出率为１００％。其它对照病毒及传代细胞ＰＣＲ反应产物电泳结果均为阴性。     

Granulocytic Ehrlichiosis in Dogs from North Carolina and Virginia

Medical records of 3 dogs from North Carolina and 3 dogs from Virginia with ehrlichial morulae in circulating neutrophils were studied retrospectively. Two clinically distinct disease syndromes, including chronic, moderate to severe anemia (n = 3) and polyarthritis (n = 2) were associated with canine granulocytic ehrlichiosis (CGE) in these dogs. One dog was clinically healthy, and abnormalities were not detected during physical examination. Clinical signs were nonspecific and included fever, lethargy, anorexia, vomiting, and diarrhea. The most frequent laboratory abnormalities were normocytic normochromic nonregenerative anemia, moderate thrombocytopenia with large platelets, lymphopenia, and eosinopenia. Considerable variability was found in the serologic responses to Ehrlichia equi, Ehrlichia canis , and Ehrlichia chaffeensis antigens among the 5 dogs for which stored sera were available for indirect fluorescent antibody testing. Polymerase chain reaction amplification and sequencing of portions of the 16S rRNA gene from blood (collected in ethylenediaminetetraacetic acid) of 1 severely anemic dog (dog 3) and 1 polyarthritic dog (dog 4) resulted in DNA sequences nearly identical to the GenBank accessions for Ehrlichia ewingii. The DNA sequence from a 3rd dog (dog 5) was most similar to that of E. canis. Serologic or molecular results support the possibility of E. ewingii, E. equi , and E. canis coinfection or serologic cross-reactivity among canine granulocytic and monocytic Ehrlichia species in dogs from North Carolina and Virginia. Variability in response to tetracycline or doxycycline treatment was noted in these dogs, with more rapid resolution of signs in dogs with polyarthritis. We report the 1st cases of CGE in dogs from North Carolina and Virginia, including recognition of CGE in a healthy dog.     

禽流感RT-PCR诊断法的建立

根据禽流感病毒（ＡＩＶ）Ａ／Ｃｈｉｃｋｅｎ／Ｂｒｅｓｌｉｎ／１９０２（Ｈ７Ｎ７）株的血凝素（ＨＡ）基因参考序列设计合成一对Ｈ７ＨＡ特异引物,并应用ＳＤＳ－蛋白酶Ｋ法提取病毒ＲＮＡ,建立了逆转录－聚合酶链式反应（ＲＴ－ＰＣＲ）检测ＡＩＶＲＮＡ的方法。应用此法对鸡胚培养的ＡＩＶ尿囊液、ＳＰＦ感染鸡粪便进行了检测,并与琼脂扩散试验和电镜技术作了对比。特异性试验以新城疫病毒（ＮＤＶ）、传染性法氏囊病病毒（ＩＢＤＶ）、传染性支气管炎病毒（ＩＢＶ）、减蛋综合征病毒（ＥＤＳ－７６Ｖ）等的感染鸡胚尿囊液作为对照。结果表明,ＡＩＶ尿囊液ＲＴ－ＰＣＲ全部阳性,ＳＰＦ感染鸡粪便８７份,ＲＴ－ＰＣＲ检出６９份阳性,样品处理浓缩后琼脂扩散检出１１份,电镜检测了２３份样品,仅检出２份阳性。非特异性对照样品经ＲＴ－ＰＣＲ检测全部阴性。将ＡＩＶ感染鸡胚尿囊液作１０倍系列连续稀释,可检出稀释度为１０－２的病毒尿囊液。ＲＴ－ＰＣＲ最早检出时间为感染后第３天,而琼扩和电镜均是７天。该法具有高度的特异性和灵敏性,且节约时间（只需８小时左右）,可从分子水平上对ＡＩＶ进行早期快速诊断和流行病学调查     

Cerebrospinal Fluid PCR and Antibody Concentrations against Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato in Dogs with Neurological Signs

Background: The tick-borne bacteria Borrelia burgdorferi sensu lato (sl) and Anaplasma phagocytophilum have been suspected to cause neurological signs in dogs. Diagnosis often has been made based on positive antibody titers in serum of dogs with neurological signs, but a high seroprevalence in dogs in at-risk populations makes diagnosis difficult.
Objective: To determine if the neurological signs in dogs examined were caused by any of these bacteria.
Animals: Fifty-four dogs presented to a board-certified neurologist.
Methods: Prospective study. We divided dogs into 2 groups: those with inflammatory diseases of the central nervous system (CNS) and those with neurological signs from other diseases. Blood and cerebrospinal fluid (CSF) from all dogs were analyzed.
Results: Dogs with inflammatory CNS diseases showed no serum antibodies against any of the agents. Among dogs with neurological signs from other diseases, 10.3% had serum antibodies for B. burgdorferi sl and 20.5% for A. phagocytophilum . All blood samples analyzed for bacterial deoxyribonucleic acid (DNA) and all CSF analyzed for antibodies and bacterial DNA for the 2 agents were negative.
Conclusions and Clinical Importance: Based on this study, these bacteria are unlikely causes of neurologic disease in dogs and the presence of serum antibodies alone does not document or establish a definitive diagnosis of CNS disease caused by these organisms. Dogs that have neurologic disease and corresponding serum antibodies against these agents should have additional tests performed to assess for other potential etiologies of the signs.     

Molecular Evidence for Persistence of Anaplasma phagocytophilum in the Absence of Clinical Abnormalities in Horses after Recovery from Acute Experimental Infection

Background: Anaplasma phagocytophilum infects several mammalian species, and can persist in sheep, dogs, and calves. However, whether this organism persists in horses or induces long-term clinical abnormalities is not known.
Objectives: To evaluate whether A. phagocytophilum can persist in horses and to document clinical findings for 3 months after complete recovery from acute disease.
Animals: Five clinically normal adult horses that had recovered spontaneously from experimentally induced acute disease caused by a Swedish equine isolate of A. phagocytophilum .
Methods: Horses were monitored for up to 129 days post inoculation (PI) by daily clinical examination and at least alternate day blood sampling for evidence of A. phagocytophilum on polymerase chain reaction (PCR) and blood smears. All horses were euthanized and underwent postmortem examination.
Results: All horses were periodically PCR positive after recovery from acute infection. Before day 66 PI 2 horses were persistently PCR negative whereas 3 horses were intermittently PCR positive. Subsequently, 4 of 5 horses were intermittently PCR positive, particularly after stress mimicking interventions. One animal was positive immediately before postmortem examination. Clinical abnormalities related to persistence of anaplasma were not observed. No specific changes were found at postmortem examination, and all sampled tissues from all horses were negative on PCR for A. phagocytophilum .
Conclusions and Clinical Importance: Infection with A. phagocytophilum can persist in the horse for at least 129 days. However, the continued presence of the organism is not associated with detectable clinical or pathological abnormalities.     

Diagnostic Features of Clinical Neurologic Feline Infectious Peritonitis

Feline infectious peritonitis (FIP) is a fatal Arthus-type immune response of cats to infection with FIP virus, a mutant of the ubiquitous feline enteric coronavirus (FECV). The disease may occur systemically or in any single organ system, and primary neurologic disease is a common subset of such manifestations. We examined 16 domestic cats with clinical neurologic FIP and 8 control cats with nonneurologic FIP, with the intention of identifying the ante-and postmortem diagnostic tests that most contribute to accurate diagnosis. Of the 16 cats with neurologic FIP, 15 were less than 2 years of age and all 16 originated from large multiplecat households. The most useful antemortem indicators of disease were positive anti-coronavirus IgG titer in cerebrospinal fluid, high serum total protein concentration, and findings on magnetic resonance imaging suggesting periventricular contrast enhancement, ventricular dilatation, and hydrocephalus. Postmortem diagnosis was facilitated by FIP monoclonal antibody staining of affected tissue and coronavirus-specific polymerase chain reaction. Most cats with neurologic and ocular forms of FIP had patchy, focal lesions, suggesting that recently developed technologies described in this report may be useful for evaluation of cats with suspected FIP.     

Genital lesions and distribution of amastigotes in bitches naturally infected with Leishmania chagasi

Recent reports indicate that Leishmania chagasi has tropism to the male canine genital system, which is associated with shedding of the organism in the semen, supporting the hypothesis of venereal transmission. The aim of this study was to describe the lesions and assess parasite load in the genital system of bitches with canine visceral leishmaniasis (CanL). Symptomatic (n=5) and asymptomatic (n=5) bitches seropositive for CanL were randomly selected at the Center for Zoonosis Control (Belo Horizonte, State of Minas Gerais, Brazil). Five serologically negative, healthy, adult bitches also from the CZC were used as controls. Samples from genital organs (vulva, vagina, cervix, uterine body, uterine horns, uterine tubes, and ovaries), liver, and spleen were histologically evaluated and processed for immunodetection of Leishmania sp., and PCR. The most significant histological change was a mild to moderate vulvar dermatitis, characterized by a histio-plasma-lymphocytic infiltrate. This change was detected in all asymptomatic, four symptomatic, and three uninfected control bitches. In one symptomatic and one asymptomatic bitch intracytoplasmic amastigotes were observed within macrophages in the inflammatory infiltrate. Samples from all the segments of the genital tract were positive in at least one infected animal, in the absence of detectable amastigotes in the tissue. These findings support the notion that L. chagasi does not have genital tropism in the bitch, which is in contrast to our previous findings in naturally infected male intact dogs.     

Detection of BHV-1 in a Naturally Infected Bovine Fetus by a Nested PCR assay

Bovine herpesvirus 1 (BHV-1) is frequently associated with abortion in naturally and experimentally infected cattle. Most of the virus isolation and immunofluorescent antibody protocols described in the literature for detecting BHV-1 in bovine foetuses are rather laborious, costly and time-consuming. The detection is described of BHV-1 in the tissues of a naturally aborted bovine foetus by a nested PCR assay with no further hybridization procedures.Optimal results were achieved by filtering the foetal tissues on a chromatography column before DNA extraction, by using two pairs of primers in a nested PCR and by evaluating the amplification products on silver-stained polyacrylamide gels.This nested PCR was faster and easier to perform than the virus isolation test. To our knowledge, this is the first time that BHV-1 has been detected in the tissues of a naturally infected bovine foetus by means of a nested PCR. The test seems to be a practical alternative for rapid detection of BHV-1 in bovine foetus.     

聚合酶链反应（PCR）鉴定猪肉方法的建立及应用

依据猪小卫星ＤＮＡ序列设计了１对引物,建立了ＰＣＲ鉴定生、熟猪肉的方法。应用本方法特异地扩增出预期的猪３０７ｂｐ的ＤＮＡ片段,其检测敏感度对生猪肉达３８．６ｆｇＤＮＡ,对熟猪肉和高压猪肉为０．３７５ｆｇ全基因组ＤＮＡ。运用该引物仅可扩增生猪ＤＮＡ的单一片段,而对马、牛、羊等１２种动物肉的ＤＮＡ扩增则呈阴性。应用本法对６３份生、熟猪肉进行鉴定,正确率为１００％,对各种样品检测,均可在６ｈ内完成。     

The role of dogs as reservoirs of Leishmania parasites, with emphasis on Leishmania (Leishmania) infantum and Leishmania (Viannia) braziliensis

Leishmania parasites cause a group of diseases collectively known as leishmaniases. The primary hosts of Leishmania are sylvatic mammals of several orders (Rodentia, Marsupialia, Carnivora, etc.). Under certain circumstances, particularly in peridomestic and domestic transmission foci, synanthropic and domestic animals can act as source of infection for phlebotomine sand fly vectors. Dogs have long been implicated as the main domestic reservoirs of Leishmania (Leishmania) infantum, the aetiological agent of zoonotic visceral leishmaniasis, and there exists an increasing trend to regard dogs as the main domestic reservoirs of Leishmania (Viannia) braziliensis, the most widespread aetiological agent of American tegumentary leishmaniasis. However, insights derived from recent research indicate that not dogs but humans are probably the most important domestic reservoirs of L. (V.) braziliensis. In the present article, the role of dogs as reservoirs of Leishmania parasites, with emphasis on L. (L.) infantum and L. (V.) braziliensis, is reviewed.     

Comparison of PCR methods for diagnosis of canine visceral leishmaniasis in conjunctival swab samples

Four PCR assays for detection of Leishmania DNA in conjunctival swab samples were compared. All methods had two steps: a first amplification followed by hybridization or by a new amplification (nested or seminested). Two methods (kDNA PCR-hybridization and kDNA snPCR) used primers targeted to the minicircles of kinetoplast DNA (kDNA) and the other two methods to the coding (LnPCR) and intergenic noncoding regions (ITS-1 nPCR) of ribosomal rRNA genes. kDNA PCR-hybridization was positive for 22/23 dogs (95.6%) and for 40/46 samples (86.9%), considering the right and the left conjunctivas. kDNA snPCR was positive for 21/23 dogs (91.3%) and for 40/46 samples (86.9%). The ITS-1 nPCR and LnPCR were both able to detect the parasites in 17/23 dogs (73.9%) and 29/46 (63%) and 30/46 (65.2%) samples, respectively. The positivities of the kDNA based methods were significantly higher; however the choice of the best method will depend on the kind of information required with the diagnosis.     

Urinary and Plasma Catecholamines and Metanephrines in Dogs with Pheochromocytoma,Hypercortisolism, Nonadrenal Disease and in Healthy Dogs

Background

Diagnosis of pheochromocytoma (PC) is based on a combination of clinical suspicion, finding an adrenal mass, increased plasma, and urine concentrations of catecholamine metabolites and is finally confirmed with histopathology. In human medicine, it is controversial whether biochemically testing plasma is superior to testing urine.

Objectives

To measure urinary and plasma catecholamines and metanephrines in healthy dogs, dogs with PC, hypercortisolism (HC), and nonadrenal diseases (NAD) and to determine the test with the best diagnostic performance for dogs with PC.

Animals

Seven PC dogs, 10 dogs with HC, 14 dogs with NAD, 10 healthy dogs.

Methods

Prospective diagnostic clinical study. Urine and heparin plasma samples were collected and stored at −80°C before analysis using high‐pressure liquid chromatography (HPLC) coupled to electrochemical detection or tandem mass spectrometry were performed. Urinary variables were expressed as ratios to urinary creatinine concentration.

Results

Dogs with PC had significantly higher urinary normetanephrine and metanephrine : creatinine ratios and significantly higher plasma‐total and free normetanephrine and plasma‐free metanephrine concentrations compared to the 3 other groups. There were no overlapping results of urinary normetanephrine concentrations between PC and all other groups, and only one PC dog with a plasma normetanephrine concentration in the range of the dogs with HC and NAD disease. Performances of total and free plasma variables were similar. Overlap of epinephrine and norepinephrine results between the groups was large with both urine and plasma.

Conclusion and clinical importance

Measurement of normetanephrine is the preferred biochemical test for PC and urine was superior to plasma.