Prevalence of hemoplasma infection among cattle in the western part of Japan

We have examined for hemoplasma infection among cattle in the Hiroshima and Miyazaki prefectures by using a sensitive real-time PCR, with SYBR Green I and with melting curve analysis, which allow to distinguish the two bovine hemoplasma species, Mycoplasma wenyonii and 'Candidatus M. haemobos'. We found 69.4% of 36 cattle in Hiroshima and 93.8% of 32 cattle in Miyazaki infected with either of these two hemoplasma species. High morbidity in western part of Japan may reflect the activity of arthropod vectors for hemoplasma transmission. We also demonstrated neonatal calves less than three months old affected with hemoplasmas without grazing in summer, suggesting a possibility of vertical transmission.     

Fatal bovine anaplasmosis in a herd with new genotypes of Anaplasma marginale, Anaplasma ovis and concurrent haemoplasmosis

Haematological and molecular analysis of blood samples was carried out during an outbreak of bovine anaplasmosis in Hungary. Acute disease was observed in five animals, two of which died. Anaplasma-carrier state was diagnosed in 69 (92%) of cattle. Further evaluation of 24 blood samples revealed concurrent infections with Mycoplasma wenyonii and 'CandidatusM. haemobos' in 22 and 21 animals, respectively. In addition, two cows were identified with rickettsaemia. Regarding molecular investigation of potential hard tick vectors, Haemaphysalis inermis and Dermacentor marginatus males collected from the animals were PCR-negative. However, in one pool (out of 18) of Ixodesricinus males, and in six pools (out of 18) of D. reticulatus males the msp4 gene of Anaplasma marginale was detected. In the same I. ricinus pool Anaplasma ovis was also identified. All ticks were negative for haemoplasmas. Anaplasma sequences yielded 97-99% homology to sequences deposited in the Genbank. This is the first report of fatal bovine anaplasmosis associated with divergent A. marginale genotypes and concurrent 'CandidatusM. haemobos' infection, as well as of an A. ovis strain in ticks collected from cattle.     

Molecular detection of Mycoplasma wenyonii and 'Candidatus Mycoplasma haemobos' in cattle in Hokkaido, Japan

Blood samples from 78 cattle were tested for hemoplasma infection using molecular methods. PCR and sequence analysis revealed that 17 cattle were infected with Mycoplasma wenyonii, while 13 were infected with 'Candidatus Mycoplasma haemobos'. Four animals were infected with both species. This is the first study to report hemoplasma species infection among cattle in Japan.     

First molecular identification of Mycoplasma ovis and 'Candidatus M. haemoovis' from goat, with lack of haemoplasma PCR-positivity in lice

In order to investigate haemotropic Mycoplasma (formerly Eperythrozoon) infection of goats, blood samples and blood-sucking lice (Linognathus stenopsis) were collected in two goat herds. DNA was extracted from 20 blood samples and from 49 lice allocated to six pools according to host individuals. Haemoplasma infection was detected in four goats by real-time PCR. From the sample with the highest bacterial load the simultaneous presence of M. ovis and 'Candidatus M. haemoovis' was demonstrated by cloning and sequencing. Louse pools were haemoplasma negative, including those from bacteraemic animals. However, not only were Anaplasma inclusion bodies seen in blood smears from goats, but relevant PCR-positivity was also detected among lice. This is the first report of a molecular investigation on caprine haemoplasmas, including analysis of their bloodsucking lice. In summary, goats are susceptible to both molecularly characterised ovine haemoplasmas. On the other hand, goat sucking lice (L. stenopsis) do not appear to be potential vectors of these agents.     

Evaluation of the stable fly (Stomoxys calcitrans) as a vector of enzootic bovine leukosis

Experiments reported here were directed at 2 questions: (1) Can the stable fly (Stomoxys calcitrans) transmit enzootic bovine leukosis? (2) Could early viremia augment the probability of transmission by this insect? In one vector experiment, calves and bovine leukemia virus (BLV)-infected cows were housed with and without stable flies. The calves were monitored serologically during a 3-month postexposure period, using the agar gel immunodiffusion test. All fly-infested and fly-free calves remained BLV-seronegative. For a second vector experiment, donor calves, newly injected with blood from BLV-infected cows with high virus expression, were tethered alternately between uninoculated, weaned BLV-seronegative calves. These groups were housed with or without flies in 2 replicate trials. The inoculated calves from the first replicate seroconverted at 16 and 23 days after inoculation; the inoculated calves from the second replicate seroconverted at 11, 16, 16, and 37 days after inoculation. All uninoculated calves remained BLV-seronegative. In a manual transmission experiment, 50 unfed stable flies were allowed to complete a meal on each of 3 BLV-seronegative calves after feeding on a BLV-seropositive cow with high (42%) virus expression. One control calf was injected with blood from the cow. Seroconversion occurred in the control calf and 1 calf on which flies were given access. A scanning electron microscopic study was made of the everted and closed mouth parts of the stable fly. Given the lymphocyte count in blood from the cow used in the manual vector transmission experiment, it was calculated that 3,950 mouth part volumes would be necessary to transmit BLV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of transmission of bovine viral diarrhea virus (BVDV) between persistently infected and naive cattle by the horn fly <Emphasis Type="Italic">(Haematobia irritans)</Emphasis>

Identifying reservoirs and transmission routes for bovine viral diarrhea virus (BVDV) are important in developing biosecurity programs. The aim of this study was to evaluate BVDV transmission by the hematophagous horn fly (Haematobia irritans). Flies collected from four persistently infected cattle were placed in fly cages attached to principal (n?=?4) and control (n?=?4) BVDV-naïve calves housed individually in isolation rooms. Flies were able to feed on principal calves, but a barrier prevented fly feeding from control calves. Flies were tested for BVDV by RT-PCR and virus isolation at time of collection from PI cattle and after 48 h of exposure on BVDV-naïve calves. Blood samples were collected from calves and tested for BVDV infection. Virus was isolated from fly homogenates at collection from PI animals and at removal from control and principal calves. All calves remained negative for BVDV by virus isolation and serology throughout the study. Bovine viral diarrhea virus may be detected in horn flies collected from PI cattle, but horn flies do not appear to be an important vector for BVDV transmission.     

Use of real-time PCR to detect Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum' in the saliva and salivary glands of haemoplasma-infected cats

Feline haemoplasma infection can cause haemolytic anaemia. The natural method of transmission of haemoplasmas between cats is currently unknown but the nature of some of the risk factors for infection suggests that saliva may act as a mode of transmission. The aim of this study was to determine if Mycoplasma haemofelis (Mhf) and 'Candidatus Mycoplasma haemominutum' (CMhm) DNAs could be amplified from saliva and salivary gland samples collected from haemoplasma-infected cats.     

Examination of the 16S-23S rRNA intergenic spacer sequences of 'Candidatus Mycoplasma haemobos' and Mycoplasma haemofelis

The intergenic spacer region between the 16S and 23S rRNA genes of mycoplasmas has been used for a genetic marker for identification of the species. Here we show the intergenic spacer regions of two hemotropic mycoplasmas, Mycoplasma haemofelis and 'Candidatus Mycoplasma haemobos (synonym: 'C. M. haemobovis')' are also useful for classification of this particular group of mycoplasms. The spacer region of M. haemofelis and `C. M. haemobos' consisted of 209 and 210 base pairs, respectively, and both lacked the spacer tRNA genes. Phylogenetic analysis suggested a monophyletic relationship among hemoplasmas and M. fastidiosum. A hypothetical secondary structure predicted in the spacer regions tentatively assigned the boxA and boxB motifs peculiar to the members of the genus Mycoplasma. M. haemofelis and 'C. M. haemobos' possessed a stem-loop structure in common, despite the presence of a palindromic nucleotide substitution in the stem region.     

Diagnosis of feline haemoplasma infection using a real-time PCR assay

Haemobartonella felis has been reclassified within the genus Mycoplasma as Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum', collectively referred to as the feline haemoplasmas. A total of 78 cats from the Johannesburg area that had blood samples submitted to a private veterinary laboratory were tested using a real-time polymerase chain reaction (PCR) assay able to detect and distinguish the two feline haemoplasma (basonym Haemobartonella) species. All samples had been diagnosed with haemoplasma infection by cytological examination of blood smears. Statistical analysis was performed to evaluate associations between haemoplasma status, age, and haematological and biochemical parameters. On PCR assay 43 cats (55%) were haemoplasma negative, 25 (32.1%) positive for 'Candidatus Mycoplasma haemominutum', 5 (6.4%) positive for Mycoplasma haemofelis and 5 (6.4%) positive for both species. Significant inverse correlation was found between the amount of M. haemofelis DNA present in the blood and the haematocrit value. Cats that were positive for M. haemofelis showed macrocytic regenerative anaemia, monocytosis and thrombocytopaenia. This report documents the existence of both haemoplasma species in cats in South Africa.     

Infectious bovine keratoconjunctivitis: contact transmission

The transmission of Moraxella bovis was studied in calves in the absence of the face fly (Musca autumnalis) or environmental conditions that might insult the eye. Thirty calves were placed in 10 groups of 1 experimentally infected calf and 2 contact calves each. Over 40 days, only 1 eye in 1 contact calf developed clinical infectious bovine keratoconjunctivitis. The organism was recovered in only 8 of 20 contact calves, whereas infection and disease occurred in all experimentally infected calves. Transmission of M bovis occurring in the absence of some other intervening factor was probably of minimal importance. Seemingly, herd preventive treatment would be most effective when flies and environmental factors are at a minimum.     

Feline hemoplasmas in Switzerland: identification of a novel species, diagnosis, prevalence, and clinical importance

Two feline hemotropic mycoplasma spp. (aka hemoplasma) have previously been recognized. We recently discovered a third novel species in a cat with hemolytic anemia, designated 'Candidatus Mycoplasma turicensis', which is closely related to rodent haemoplasmas. This novel species induced anemia after experimental transmission to two SPF cats. Three quantitative real-time PCR assays were newly designed and applied to an epidemiological study surveying the Swiss pet cat population. Blood samples from 713 healthy and ill cats were analyzed. Up to 104 parameters per cat (detailed questionnaire, case history, laboratory parameters and retroviral infections) were evaluated. 'Candidatus Mycoplasma haemominutum' infection was more prevalent (8.5%) than Mycoplasma haemofelis (0.5%) and 'Candidatus Mycoplasma turicensis' (1%). Hemoplasma infections were associated with male gender, outdoor access, and old age, but not with disease or anemia. Infections were more frequently found in the South and West of Switzerland. Several hemoplasma infected cats, some acutely infected, others co-infected with FIV or FeLV, showed hemolytic anemia indicating that additional factors might play a role in the pathogenesis of the disease.     

Confirmation of vertical transmission of bovine immunodeficiency virus in naturally infected dairy cattle using the polymerase chain reaction.

The purpose of this study was to determine whether bovine immunodeficiency virus (BIV) is vertically transmitted in naturally infected dairy cattle. Twenty-two dam/calf pairs from a Mississippi Agriculture and Forestry Experiment Station dairy were the study group. Blood samples were collected following delivery of calves, the peripheral blood leukocytes were purified from these samples, and the leukocyte DNA was used in polymerase chain reactions targeting the pol gene region of the BIV provirus. Southern blotting and hybridization were used to confirm the BIV specificity of the amplified fragments. BIV provirus was detected in 14 of 22 calves (64%), demonstrating vertical transmission. Eight of the calves were disqualified from the final interpretation of transplacental transfer because they may have nursed their mothers prior to blood collection, allowing the possibility of lactogenic transfer of the virus. Transplacental transmission of BIV was identified in 6 of 22 calves (27%).     

Occurrence and genetic diversity of hemoplasmas in beef cattle from the Brazilian Pantanal,an endemic area for bovine trypanosomiasis in South America

Hemotropic mycoplasmas (hemoplasmas) are Gram-negative bacteria that parasitize the erythrocyte surface of a wide variety of mammals. The present study aimed at investigating the occurrence of hemoplasmas in beef cattle in the Brazilian Pantanal, an area endemic for bovine trypanosomiasis in South America. Additionally, the objective of this study was to characterize molecularly the genotypes of the found hemoplasmas. For this purpose, blood and serum samples of 400 beef cattle were collected from five properties in Corumbá, Nhecolândia sub-region, Mato Grosso do Sul, in Midwest Brazil. Blood samples underwent DNA extraction and standard 16S rRNA gene-based PCR assays for hemoplasmas. The sequences obtained were submitted to phylogenetic inferences, distance analysis, and genotype diversity. The Indirect Enzyme-Linked Immunoabsorbent Assay (iELISA) indicated the presence of anti-Trypanosoma vivax IgG antibodies in 89.75% of the animals sampled, confirming the endemicity of said agent in the studied region. Among the 400 bovine blood samples tested, 2.25% (9/400) were positive for hemoplasmas in cPCR. The phylogenetic analysis of the obtained sequences confirmed the presence of 'Candidatus Mycoplasma haemobos' and Mycoplasma wenyonii DNA in 0.5% (2/400) and 1.75% (7/400) animals, respectively. Five genotypes of M. wenyonii and one of 'Candidatus M. haemobos' were detected among the sequenced amplicons. The present study showed low molecular occurrence of haemoplasmas in beef cattle sampled in the Brazilian Pantanal, an area endemic for bovine trypanosomiasis. Despite of the conservation of the 16S rRNA gene, there was considerable diversity of hemoplasma genotypes infecting the sampled beef cattle.     

Transplacental and oral transmission of wild-type bluetongue virus serotype 8 in cattle after experimental infection

Potential vertical transmission of wild-type bluetongue virus serotype 8 (BTV-8) in cattle was explored in this experiment. We demonstrated transplacental transmission of wild-type BTV-8 in one calf and oral infection with BTV-8 in another calf. Following the experimental BTV-8 infection of seven out of fifteen multi-parous cows eight months in gestation, each newborn calf was tested prior to colostrum intake for transplacental transmission of BTV by RRT-PCR. If transplacental transmission was not established the calves were fed colostrum from infected dams or colostrum from non-infected dams spiked with BTV-8 containing blood. One calf from an infected dam was born RRT-PCR positive and BTV-specific antibody (Abs) negative, BTV was isolated from its blood. It was born with clinical signs resembling bluetongue and lived for two days. Its post-mortem tissue suspensions were RRT-PCR positive. Of the seven calves fed colostrum from infected dams, none became infected. Of the six calves fed colostrum from non-infected dams spiked with infected blood, one calf became PCR-positive at day 8 post-partum (dpp), seroconverted 27 days later, and remained RRT-PCR and Abs positive for the duration of the experiment (i.e., 70 dpp). This work demonstrates that transplacental transmission in late gestation and oral infection of the neonate with wild-type BTV-8 is possible in cattle under experimental conditions.     

The detection of Mycoplasma (formerly Eperythrozoon) wenyonii by 16S rDNA PCR and denaturing gradient gel electrophoresis

Although the role of Mycoplasma wenyonii in disease is still subject to some debate, infections have been reported to result in parasitaemia, anaemia, scrotal and hind limb oedema, tachycardia, pyrexia, infertility, swollen teats, prefemoral lymphadenopathy and decreased milk production. Previously, diagnosis of M. wenyonii has been based on blood smears but is not specific for M. wenyonii and can be difficult to interpret. We have previously described the use of PCR and denaturing gradient gel electrophoresis (DGGE) for the detection and differentiation of Mycoplasma species. DGGE enables the rapid and specific identification of Mycoplasma species and is ideally suited to detecting both mixed infections and new and unusual species. In this study, we have used DGGE with universal primers to detect M. wenyonii DNA from blood samples. DGGE can be used on blood samples as a rapid and specific test for M. wenyonii and can also be used as a screening test for other blood borne pathogens.     

Occurrence of 'Candidatus Mycoplasma turicensis' infection in domestic cats in Japan

We have examined the presence of hemoplasmas, hemotropic mycoplasmas, among domestic cats (Felis catus) in Japan by using a species specific PCR, and found 'Candidatus Mycoplasma turicensis', a recently recognized hemoplasma species. A total of 60 feline blood samples collected in 2004 and 2005 were subjected to PCR amplification for the detection of Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum' and 'Candidatus Mycoplasma turicensis'. Six blood samples collected from domestic cats were found infected with the 'Candidatus M. turicensis'. All of them are also infected with other species of hemoplasmas, M. haemofelis and/or 'Candidatus M. haemominutum'. This is the first to demonstrate 'Candidatus M. turicensis' infections among cat population in Japan.     

Role of insects in the transmission of bovine leukosis virus: potential for transmission by stable flies, horn flies, and tabanids

The ability of stable flies (Stomoxys calcitrans), horn flies (Haematobia irritans), and tabanids (Diptera: Tabanidae) to transmit bovine leukosis virus (BLV) was investigated. Stable flies and horn flies were fed on blood collected from an infected cow, and the flies' mouthparts were immediately removed, placed in RPMI-1640 medium, ground, and inoculated into sheep and calves. Infection of sheep occurred with mouthparts from as few as 25 stable flies or 25 horn flies. However, sheep were not infected when removal of stable fly mouthparts was delayed greater than or equal to 1 hour after blood feeding. Infection of calves occurred after inoculation of mouthparts removed immediately after feeding from as few as 50 stable flies or 100 horn flies. Infected blood, applied by capillary action to the mouthparts (labella) of 15 deer flies (Chrysops sp) and a single horse fly (Tabanus atratus) caused infection in each of 2 sheep. Infection did not occur in 2 calves inoculated daily for 5 days with mouthparts from 50 horn flies collected after feeding on a BLV-infected steer. Four calves receiving bites from 75 stable flies interrupted from feeding on a BLV-positive cow also were not infected. Seronegative cattle held for 1 to 4 months in a screened enclosure with positive cattle in the presence of biting flies were not infected with BLV. The feeding behavior of each insect is discussed to assess their potential as vectors of BLV.     

The epidemiology of Parafilaria bovicola in the Transvaal Bushveld of South Africa

A total of 20 375 flies collected off cattle on 12 farms over 36 months were identified and examined for 3rd stage P. bovicola. The 3 vector species accounted for 64.1% of the flies collected and were the only fly species found to be infected. Musca lusoria was clearly the dominant vector fly, although large numbers of Musca sp. A appeared regularly between February and April each year. This phenomenon, coupled with high numbers of M. lusoria throughout most of the year, led to an increase in the numbers of vector flies from their lowest level in June to a peak in February-April. Of the 13 070 vector flies examined for 3rd stage larvae only 64 (0.52%) were positive; of these 41 were M. lusoria and 17 Musca sp. A. No positive male flies were found. Incubation of wild-caught flies for up to 13 days at 27 degrees C noticeably increased the larval recovery rate. Flies were found to be infected mainly from August-March. Infected M. lusoria were recorded from July-March and infected Musca sp. A from January-May. Only 6 infected M. xanthomelas were collected and this was during the period August-December, when most ovipositional blood spots occur on cattle. It is concluded that P. bovicola transmission in the Bushveld is not correlated with peak periods of bleeding but rather with high numbers of vector flies, the various species augmenting each other so that transmission may take place almost throughout the year.     

Coccidia in cows and calves on pasture in north Georgia (U.S.A.)

The prevalence and abundance of coccidian oocysts were determined in a herd of beef cows and calves on fescue pastures in the Piedmont area of Georgia during 4-consecutive grazing seasons. Twelve species of Eimeria were found in the feces of the calves and 10 species were found in the feces of the cows. Eimeria bovis was the most prevalent species found in both the calves and cows. It occurred in 72.5% of 1090 samples from the calves and 10.2% of 719 samples from cows. Eimeria bovis also comprised the majority of oocysts present in the fecal samples from the calves and cows. The greatest number of E. bovis in a sample was 45 800 oocysts per gram of feces (OPG) from a calf and 1900 OPG from a cow. No cases of clinical coccidiosis were seen in any of the animals sampled during the survey.     

A novel haemoplasma species identified in archived primate blood smears

In order to confirm a microscopic diagnosis of 'eperythrozoonosis' made over 40 years ago in a captive owl monkey (Aotus trivirgatus), DNA was extracted from archived fixed and stained blood smears and subjected to generic haemotropic mycoplasma (haemoplasma) quantitative real-time PCR (qPCR) and a human glyceraldehyde-3-phosphate dehydrogenase qPCR as an amplification control. The qPCRs confirmed the extraction of host DNA from the samples and the presence of a haemoplasma species. Partial 16S rRNA and ribonuclease P ribosomal gene fragments were amplified by PCR, cloned and sequenced. Sequence data and phylogeny showed the owl monkey haemoplasma to lie in the haemominutum clade of haemoplasmas, most closely related to 'Candidatus Mycoplasma kahaneii'. This study confirms the use of generic haemoplasma qPCRs to successfully amplify haemoplasma DNA from fixed, stained and archived blood smears from the early 1970s and provides molecular confirmation of the existence of a novel haemoplasma species in an owl monkey, for which the name 'Candidatus Mycoplasma aoti' sp. nov. is proposed.