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Photomicrograph of cerebrospinal fluid cytology. Mixed cell pleocytosis composed of mononuclear cells, lymphocytes, and neutrophil. Several round-shaped basophilic intracytoplasmic inclusions were seen in mononuclear cells (Wright-Giemsa stain).
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... Te amplifcation of the hemoplasma 16S rRNA gene was carried out using a broad-range nested PCR approach, employing primers previously described by Kaewmongkol Veterinary Medicine International et al. [17][18][19]. Te primary PCR utilized V1-F and V9-R primers, while the secondary PCR employed V3-F and V6-R primers. Te specifc oligonucleotide sequences and thermal cycle conditions for both the primary and nested PCR are provided in Table 1. ...
Hemotropic mycoplasmas, also known as hemoplasmas, are parasitic bacteria that infect red blood cells, potentially leading to varying degrees of anemia across numerous mammalian species, including nonhuman primates. The present study aims to investigate the prevalence of hemoplasma infection and identify the species involved among free-ranging Assamese macaques (Macaca assamensis) inhabiting northern Thailand. A total of 133 blood samples were collected from Assamese macaques in Chiang Rai province, Thailand, and subjected to screening for hemoplasma infection utilizing nested PCR amplification targeting the 16S rRNA gene. Positive samples were subsequently analyzed through nucleotide sequencing and phylogenetic analysis for putative species identification. Current study results revealed that 17.3% (23/133; 95% CI 11.29-24.81) of Assamese macaques tested positive for hemoplasma infection using the nested PCR assay. Partial 16S rRNA sequences derived from hemoplasma isolates in Assamese macaques exhibited 99% homology, forming a cluster within the same phylogenetic clade as “Candidatus Mycoplasma haematomacacae,” previously identified in long-tailed macaques, rhesus macaques, and Japanese macaques. These findings suggest the presence of “Ca. M. haematomacacae” not only in long-tailed macaques and rhesus macaques but also in Assamese macaques in Thailand. To our knowledge, this marks the first molecular detection of “Ca. M. haematomacacae” in Assamese macaques in Thailand. These results hold significance as they enhance our understanding of hemoplasma infection distribution among macaque populations in Thailand.
... In Thailand, DNA sequence analyses of the gp36 gene have confirmed the genetic diversity of bacterial strains and elucidated geographical patterns of the local genotypes [16][17][18]. The target genes that have been used for RT-PCR are extremely diverse, and there has been no systematic evaluation of genes that are suitable for the detection of local genotypes of E. canis by RT-PCR in Thailand and Southeast Asia. ...
Background and Aim: Because of the diversity of local genotypes of Ehrlichia canis, genes targeted by TaqMan real-time polymerase chain reaction (RT-PCR) assays should be systematically evaluated. This study evaluated the amplification efficiency, linearity, precision, and sensitivity of two TaqMan RT-PCR assays targeting the dsb and gltA loci of E. canis in recombinant plasmids and naturally infected dogs.
Materials and Methods: Thirty blood samples were collected from dogs showing clinical signs of canine monocytic ehrlichiosis at the Kasetsart University Veterinary Teaching Hospital, Bangkok, Thailand. The dsb and gltA genes were amplified by conventional PCRs (cPCRs) on the blood samples and were then sequenced. Meanwhile, RT-PCR was used to detect dsb and gltA genes in 10-fold dilutions of the recombinant plasmids.
Results: Both dsb and gltA were amplified with a high degree of linearity (R2≥0.975 and 0.993, respectively) in all dilutions, although the mean percentage of relative standard deviation of gltA was lower, but the difference was non-significant. The detection limits of RT-PCR and cPCR were 10–7 and 10–6, respectively, for both loci. RT-PCR targeting dsb (22/30; 73.3%) and gltA (15/30; 50%) yielded a number of positive results that did not differ significantly (p=0.06). The RT-PCR positive results of the dsb gene (22/30) differed significantly from that of cPCR (11/30) (p=0.004). In contrast, the RT-PCR positive results of the gltA gene (15/30) did not differ significantly from that of cPCR (12/30) (p=0.43). The mean Ct value (30.2) based on dsb RT-PCR of 22 positive cases was higher than that of gltA RT-PCR (Ct=27.4) on 15 positive cases. The Ct values from dsb RT-PCR were >30 in all seven discordant samples that were not detected by the gltA RT-PCR.
Conclusion: RT-PCR targeting the dsb gene was more sensitive for detecting E. canis in naturally infected dogs. This study suggested that TaqMan RT-PCR of the dsb gene should be selected for E. canis research in this region.
... A previously described broad-range nested PCR protocol based on the 16S rRNA gene [23] was used to amplify Mycoplasma spp. DNA using two sets of oligonucleotides. ...
Background and Aim: Hemoplasmas are defined as small, epicellular parasitic bacteria that can infect the red blood cells of several mammalian species. Diseases caused by these bacteria range from asymptomatic infections to acute hemolytic anemia. However, data on hemoplasmas in non-human primates in Thailand remain to be limited. Therefore, this study aims to determine the occurrence and genetic diversity of hemoplasmas among long-tailed macaques in Thailand.
Materials and Methods: Blood samples were collected from 339 long-tailed macaques in three provinces of Thailand. DNA was then extracted from the blood samples and tested for hemoplasma using broad-range nested polymerase chain reaction (PCR) based on the 16S rRNA gene. PCR-positive samples were sequenced, and phylogenetic analysis for species identification was conducted.
Results: In total, 38 (11.2%) out of the 339 samples were found to be positive for hemoplasmas, based on the broad-range nested PCR assay of the 16S rRNA gene. The 16S rRNA sequences of Mycoplasma spp. were highly similar (98-99% identity) to "Candidatus Mycoplasma haemomacaque." Furthermore, phylogenetic analysis using maximum likelihood demonstrated that the sequences were located in the same cluster of "Ca. M. haemomacaque."
Conclusion: The detection of hemoplasmas among long-tailed macaques in Thailand is reported. Genetic characterization confirmed that these hemoplasmas are closely related to "Ca. M. haemomacaque." These results indicate that long-tailed macaques in several locations in Thailand may be infected and serve as reservoirs for this parasite.
... The demonstration of Ehrlichia spp. morulae in monocytes, macrophages and lymphocytes in stained smears from whole blood or buffy coat, and less frequently in lymph node, bone marrow, spleen, liver and cerebrospinal fluid smears, is also helpful in establishing a definitive diagnosis of acute CΜE (Mylonakis et al., 2010;Harrus et al., 2012;Kaewmongkol et al., 2016). Although, doxycycline is highly recommended in management in acute or subclinical infections, it may invariably not be effective in completely eliminating E. canis infection in some chronic cases (Fourie et al., 2015;Schulz et al. 2011). ...
... Demonstration of Ehrlichia spp. morulae in monocytes, macrophages and lymphocytes in stained smears from whole blood or buffy coat, and less frequently in lymph node, bone marrow, spleen, liver and cerebrospinal fluid smears, is also helpful in establishing a definitive diagnosis of acute canine monocytic ehrlichiosis (CΜE) (Mylonakis et al., 2010;Harrus et al., 2012;Kaewmongkol et al., 2016). Rapid improvement noticed shortly after the institution of doxycycline treatment is attributed to the acute course of ehrlichiosis in the patient. ...
An eleven-year-old Dachshund cross-bred dog was presented to the Small Animal Clinic of Ahmadu Bello University Veterinary Teaching Hospital, Zaria, with complaints of reduced appetite noticed 2 days earlier, and an enlarged testis was noticed three months prior to presentation. Clinical diagnosis was based on history of reduced appetite and enlarged testis; physical findings such as pyrexia, tick infestation, congested ocular mucous membranes, generalized enlarged superficial lymph nodes, enlargement of the mammary glands, alopecia, atrophy of the right testis and discoloration of the scrotum with large, firm palpable mass; complete blood count revealed slight anaemia (PCV 34 %, neutrophilia (12.88 x 109/L) and presence of band cells (0.32 x 109/L); buffy coat smear showed the presence of intracytoplasmic morulae in just one monocyte in the peripheral blood smear; Ehrlichia canis antibodies was detected in serum using Ehrlichia test kit (immunochromatography); serum oestradiol-17β (42.05 pg/mL) and anti- Müllerian hormone (AMH; 25.50 μg/mL) were employed as serum tumour markers; transverse and longitudinal scrotal sonographs revealed diffuse non-homogenous tissues with increase in the echogenicity of the testicles; radiographic findings of thoraco-abdomino-pelvic regions revealed normal organ opacity indicating absence of metastases. Tissue samples were taken from the tumor following surgical excision and submitted for histopathology. The findings were large cytoplasmic vacuoles with slightly irregular nucleoli. It was confirmed to be sertoli cell tumor. The conditions were managed medically, and by surgical excision of the tumour and scrotal ablation. Outcome was successful, and patient started feeding 24 hours after the surgery.
... 14 In rare cases, E canis morulae have been identified on CSF cytology samples. 7,15,16 As suggested by the term "Canine monocytotropic ehrlichiosis," morulae are expected to be seen within monocytes/macrophages and not inside granulocytes (neutrophils), as expected with Ehrlichia ewingii infection. ...
An 8‐year‐old neutered female English Pointer was referred to a veterinary referral center (southwest of England) with a 4‐5‐month history of fecal incontinence and no evidence of urinary incontinence. Blood and free‐catch urine samples were collected and sent to an off‐site laboratory. Further investigations were postponed until laboratory results were available. Blood results showed a mild leukopenia, mild nonregenerative anemia, moderate to marked thrombocytopenia, and a mild increase in ALT and ALP activities. The primary veterinarian and client did not proceed with any further investigations for thrombocytopenia. Three weeks after the initial presentation, there was considerable clinical deterioration and progression of neurologic signs. Thoracic radiographs and an abdominal ultrasonographic examination were unremarkable. Magnetic resonance imaging (MRI) of the brain and spinal cord revealed an intramedullary lesion at the level of the C7 vertebra, a cystic lesion in the forebrain, and a bilateral lesion in the thalamus. A lumbar cerebrospinal fluid (CSF) was collected. CSF analysis showed a robustly increased protein concentration and marked pleocytosis. The cytologic evaluation revealed a mixed cellular population. Occasional neutrophils and monocytoid cells showed purple spherical intracellular inclusions, resembling Ehrlichia morulae. An aliquot of CSF was used off‐label with a dot ELISA test, which showed a strong positive result for antibodies against Ehrlichia canis/Ehrlichia ewingii. PCR identified these morulae to be E canis. To best of the authors’ knowledge, this is the first case of ehrlichial infection in canine CSF where Ehrlichia sub‐species morulae present within neutrophils were confirmed to be Ehrlichia canis using PCR.
... PCR detection of E. canis was performed with PCR primers for the gp36 gene (EC36-F1 (5 0 -GTATGTTTCTTTTATATCATGGC-3 0 ) and EC36-R1 (5 0 -GGTTATATTTCAGTTATCAGAAG-3 0 )) ( Table 3). The PCR cycles and conditions used were based on previous studies Kaewmongkol et al., 2016Kaewmongkol et al., , 2017. ...
The efficacy of antibody detection tools for all stages of Ehrlichia canis infections and for various genotypes remains unclear. We produced recombinant gp36 (rgp36) antigens from different isolates of Thai E. canis to confirm the immunoreactivities to these recombinant proteins from naturally infected dogs. Sera and blood samples were taken from 21 dogs naturally infected with E. canis and in the clinical stages of acute phase ehrlichiosis. The expression vectors and competent E. coli produced two isolates of rgp36. These two major rgp36s were recognized by the dogs' sera in Western blotting, with both anti-dog IgM and IgG used as secondary antibodies. The two different genotypes of these local recombinant immunoreactive proteins were gp36 subgroup A (isolate 1055) and subgroup B (isolate 533). The Western blot analyses successfully identified both specific IgM and IgG from the dogs' sera. Of all 21 cases, five dogs presented specific IgM, twenty dogs presented specific IgG, and the commercial test used found fifteen seropositive dogs. There were four dogs that presented both specific IgM and IgG. Only one dog presented specific IgM only. This report is the first identification of a specific IgM in dogs in response to acute infections with E. canis. The recombinant gp36 isolates may be useful as potential antigenic material for subsequent serological tests that have a high possibility for differentiating between acute, chronic, primary, and nonprimary infections with E. canis.
... DNA was extracted from the contents inside the abscess using an E.Z.N.A. ® Tissue DNA Kit [15,16]. The rpoB loci were selected as the housekeeping gene targets for genetic analysis of those bacteria. ...
Background:
The Sanger sequencing technique has been questioned and challenged by advanced high-throughput sequencing approaches. Sanger sequencing seems to be an obsolete technology. However, there are still research problems that could be answered using the Sanger sequencing technology. Fastidious obligate anaerobic bacteria are mostly associated with abscesses in animals. These bacteria are difficult to isolate from abscesses and are frequently excluded due to the bias of conventional bacterial culturing.
Aim:
This study demonstrated the usefulness of a broad-range polymerase chain reaction (PCR) with Sanger sequencing to identify the majority population of bacteria in abscesses from exotic pet animals.
Materials and methods:
This study performed a pilot investigation of abscesses from 20 clinical cases (17 rabbits, 2 hedgehogs, and 1 sugar glider) using standard culture methods for both aerobes and anaerobes and broad-range nested PCR targeting the 16S rRNA gene followed by the Sanger sequencing technique.
Results:
The standard culture and PCR techniques detected bacteria in 9 and 17 of 20 samples, respectively. From the 17 sequencings of the 16S rRNA, 10 PCR products were found to be closely related with obligate anaerobes including Bacteroides spp., Fusobacterium spp., Prevotella spp. Phylogenetic analysis using the rpoB gene revealed that the species for the Bacteroides was thetaiotaomicron and for the Fusobacterium was varium and nucleatum. However, the amplification of the rpoB gene for the Prevotella spp. was unsuccessful. Correlations between the standard culture and PCR techniques were found in 9 (6 positive and 3 negative samples) of 20 samples. Eleven samples were discordant between the standard culture and PCR techniques which were composed of eight samples negative by culture but positive by PCR and three samples had different bacteria by the culture and PCR techniques.
Conclusion:
According to this study, broad-range PCR combined with Sanger sequencing might be useful for the detection of dominant anaerobic bacteria in abscesses that were overlooked based on conventional bacterial culture.
... This study revealed the presence of the A strain (Thai CM172), which contained 143 amino acids of the pre-TR region. This strain was highly similar in the pre-TR region to geographically dispersed strains from the USA, Nigeria, Brazil, Cameroon, Israel, Mexico, and Spain (Doyle et al., 2005a;Doyle et al., 2006;Kamani et al., 2013;Zweygarth et al., 2014;Kaewmongkol et al., 2016). The remaining strains (Thai CM180 and CM196), which contained 142 amino acids in the pre-TR region, were similar to strains from Taiwan and South Africa Zweygarth et al., 2014). ...
Ehrlichia canis is the causative agent of canine monocytic ehrlichiosis (CME). While there is a high prevalence of CME in Thailand, genetic diversity of E. canis is still poorly defined. This study examined the molecular characteristics of E. canis using PCR and phylogenetic analysis of the dsb, gp19 and gp36 genes. DNA was extracted from 220 whole blood samples of naturally infected dogs, and all had clinical signs compatible with tick-borne diseases. Of these, 16.4% (36/220) provided positive E. canis DNA via the dsb and gp19 genes. However, only 13 out of the 36 samples (36.1%) were positive for the gp36 gene. Sequences of the dsb gene had very high identity (99-100%) with previously deposited E. canis sequences. Sequences of the gp19 gene were similar to those from US and Taiwanese genogroups (98.8-99.5% identity). Elucidation of genetic characteristics of E. canis based on the gp36 gene displayed 91.4-99.1% shared identity. There were 426-429 bp of a 5' end pre-repeat tandem region, a 27 bp repetition with variable numbers of a tandem repeat (TR) region of 9 amino acid sequences (TEDSVSAPA), and a variable 3' end region with sequence length depending on the isolate (72-93 bp). Phylogenetic trees of E. canis, particularly using the gp36 amino acid sequences, showed that the Thai strains fell into two phylogenetic clades contained within other worldwide E. canis strains. Alignment and phylogenetic analysis suggested that E. canis strains from Thailand could be divided into two genogroups, the US and Taiwanese genogroups. This study provides the first characterization of the dsb and gp19 genes of E. canis in Thailand, the results support the conclusion that the gp36 is a potential target for genotyping and elucidation of phylogenetic relationships among E. canis strains.
... This case mainly emphasizes the cerebral involvement in Ehrlichiosis which should be ruled out whenever cases with neurological entity are presented. However, cerebral involvement in Ehrlichiosis has been much less reported [7] , but one such case with cerebral signs with normal thrombocyte count has been reported in Thailand. Blood smear examination can be helpful in the initial diagnosis but only with 4% sensitivity [8] . ...
... The above findings were similar to that of cases with cerebral form of Ehrlichiosis wherein the CSF resembles that of viral diseases (i.e., the WBC count and protein may be normal or slightly to moderately elevated with a predominantly mononuclear pleocytosis which was clearly stated in previous reports [15,16] . The above findings of nested PCR were in agreement with the previous findings [12,7] . Hence, the present study revealed the cerebral involvement of Ehrlichia canis and its further evidence through cerebrospinal fluid cytology and utilization of acridine orange staining in CSF and molecular characterization of the same. ...
A 3 year old intact female German shepherd dog with history of inappetance, pyrexia, paraplegia and uveitis was presented to the small animal outpatient unit of the Department of Clinics, Madras Veterinary College, Chennai. Haemato-biochemical values revealed relative thrombocytopenia with low hemoglobin and red blood cell count and moderate elevation in the Blood urea nitrogen. Radiographic imaging revealed mild splenomegaly. A standard cerebrospinal fluid examination was performed. Cerebrospinal fluid was slightly turbid with elevated glucose and protein levels. CSF Cytology revealed monocytic pleocytosis with several intracytoplasmic basophilic round to oval inclusion bodies in multifocal fields consistent with Ehrlichia canis morulae within the cytoplasm of the monocytes which was further confirmed with acridine orange staining method. Nested Polymerase chain reaction was done on CSF to confirm the Ehrlichiosis induced meningoencephalitis in the present case. The dog was treated with respective antibiotics. Later euthanized under humanitarian grounds upon owner’s request.
... cell/lL), and absence of thrombocytopenia.20 However, the authors did not perform a molecular investigation for toxoplasmosis and neosporosis; and therefore, neither of these infectious agents were ruled out as causative agents. ...
... The CSF total protein and TNCC in the present case were much higher, and eosinophils were not detected in the mild pleocytosis of the former case. 20 CME can cause meningitis or meningoencephalitis, which are associated with thrombocytopenia-induced meningeal bleeding in dogs.20,26,27 However, neurologic manifestations of CME are considered uncommon.12 ...
An 8-year-old mixed-breed dog was presented for acute, progressive weakness and ataxia, inappetence, and weight loss. The patient was mentally normal, but nonambulatory, with a right head tilt, right positional ventral strabismus, and slight head tremors. A neurologic lesion was localized to the cerebellum and right brainstem. Cerebrospinal fluid (CSF) analysis showed a markedly increased protein concentration and mixed pleocytosis, with eosinophil predominance (44%), intracytoplasmic inclusions within eosinophils, consistent with Ehrlichia canis (E canis) morulae, and Toxoplasma gondii (T gondii) or Neospora caninum (N caninum) tachyzoites within eosinophils and monocytes. A serum indirect immunofluorescent antibody test was positive for N caninum (titer 1:12 800) and negative for T gondii. Both blood and CSF PCR results were N caninum- and E canis-positive and T gondii- and Anaplasma phagocytophilum-negative, and blood PCR, but not CSF PCR, was Hepatozoon canis-positive. The dog was treated for 30 days with clindamycin, sulfamethoxazole-trimethoprim, doxycycline, prednisone, and cephalosporin, but did not improve neurologically, and was euthanized. Brain histopathology showed moderate multifocal, subacute meningoencephalitis with necrosis and gliosis. The neurologic disease was mostly attributed to central nervous system (CNS) neosporosis, with the possible contribution of ehrlichiosis, which was likely a manifestation of blood-brain barrier disruption. Hepatozoonosis was probably a result or cause of underlying immunosuppression. To our knowledge, this is the first report of CNSN caninum and E canis co-infection detected by both CSF PCR and cytology and E canis morulae identified within CSF eosinophils.
