A comprehensive evaluation and first molecular report of Theileria ovis infection in small ruminants in Saudi Arabia

A total of 1000 clinically healthy small ruminants comprising 500 sheep and 500 goats from five districts within Riyadh Province in Saudi Arabia were investigated by routine Giemsa staining for hematozoan parasites. Out of these, 100 sheep and 95 goat samples were investigated by PCR using three pairs of hemoprotozoan-specific primers. Based on microscopic examination, 33.2% of sheep and 25.2% of goats were found infected with hemoprotozoan parasites, while PCR detected hematozoan infection in 46% of sheep and 33.7% of goats. Extensive molecular characterization of hematozoan infection using six pairs of species-specific primers revealed the dominance of Theileria ovis, rather than any other species, which is recorded for the first time in small ruminants in Saudi Arabia. Prevalence of T. ovis in sheep and goats was found to be the highest in Riyadh (32, 48%) followed by AL-Kharj (31, 35%), Ad-Dawadimi (31, 33%), AL-Majmaah (15, 27%), and Rumah (17, 23%). The highest parasite prevalence was recorded in the 3 years of age and >?4 years of age ruminants, while the lowest prevalence was recorded in <?1 year of age ruminants. No noticeable differences in parasite prevalence between male or female ruminants were recorded. Partial sequencing of 18S rRNA gene revealed the infection of the studied ruminants with four new isolates of T. ovis. Further characterization of the pathogenicity and the clinical effects of these T. ovis isolates in sheep and goats is highly needed. The current results can be helpful in protecting and improving livestock industry in the countries that depend on a high number of small ruminants.

     

Detection of Mycoplasma conjunctivae in sheep affected with conjunctivitis and infectious keratoconjunctivitis

AIM: To determine the aetiolog y of a recurring and severe form of infectious keratoconjunctivitis (IKC) in sheep.

METHODS: Five sheep flocks that had experienced a severe form of IKC were examined. Clinical history, conjunctival swabs and blood samples were collected from affected animals. Culture for bacteria, and also specifically for Mycoplasma and Chlamydophila spp, and detection of Mycoplasma conjunctivae DNA by polymerase chain reaction (PCR) were attempted. Serum samples were tested for antibodies to M. agalactiae, M. capricolum, M. conjunctivae and Chlamydophila spp.

RESULTS: Mycoplasma conjunctivae DNA was detected using PCR in 3/5 flocks, and in all flocks antibodies to M. conjunctivae were detected in sera. A pure growth of Branhamella ovis was cultured from conjunctival swabs from a small proportion of sheep in two flocks. No other pathogens were detected.

CONCLUSIONS: This investigation demonstrated that M. conjunctivae was a primary pathogen causing severe IKC in sheep, and is the first report of detection of this organism in sheep in New Zealand. Introduction of clinically normal carrier sheep appeared to have caused the outbreaks.     

Mycoplasma ovis infection in goat farms from northeastern Brazil

Although Mycoplasma ovis (formerly Eperythrozoon ovis) has been described in small ruminants worldwide, data on M. ovis in goats remain scarce. Accordingly, the aims of the present study were to i) determine the prevalence of hemoplasmas in goats, ii) identify the tick species parasitizing the animals, and iii) determine factors associated with infection in five dairy and three beef goat farms from the Paraíba State, northeastern Brazil. Blood samples were obtained from 402 goats. Samples were screened for hemoplasmas using a pan-hemoplasma PCR. The positive samples were confirmed by sequencing. An epidemiological questionnaire was given to each farm owner addressing age, gender, and presence of ticks. A total of 158/402 (39.3%) goats were positive for M. ovis by PCR. Sequencing of PCR positive samples has shown ≥99% identity with multiple M. ovis 16S rDNA sequences deposited in GenBank, including M. ovis isolates from humans. Dairy (OR = 2.15; 95% CI: 1.40–3.32%; P = 0.0004) and anemic goats (OR = 2.33; 95% CI: 1.51–3.71%; P = 0.0001) were more likely to be infected than beef and non-anemic animals, respectively. Amblyomma parvum (49/52, 94.23%) and Rhipicephalus microplus (3/52, 5.77%) were the tick species found parasitizing the animals, with no significant association between the presence of ticks and infection by M. ovis (P = 0.1164). This is the first reportedly molecular detection of M. ovis infection in goats from South America. In conclusion, M. ovis is highly prevalent in goats from northeastern Brazil, mainly in dairy animals.     

Investigation of bovine haemoplasmas and their association with anaemia in New Zealand cattle

CASE HISTORY AND CLINICAL FINDINGS: A dairy cow, from a herd in the Waikato region of New Zealand, was reported with regenerative anaemia on 12 September 2014. Testing of blood from the animal using PCR assays for Theileria orientalis produced a negative result for both Chitose and Ikeda types.

LABORATORY FINDINGS: Using PCR and DNA sequencing, blood from the cow was positive for Candidatus Mycoplasma haemobos. Further testing of another 12 animals from the case herd, 27 days after the affected cow was first reported, showed 11 animals were positive for Candidatus M. haemobos or Mycoplasma wenyonii in the PCR. None of these cattle were clinically anaemic or positive for T. orientalis Ikeda type using PCR.

A convenience sample of 47 blood samples from cattle throughout New Zealand, submitted to the Investigation and Diagnostic Centre (Ministry for Primary Industries) for surveillance testing for T. orientalis Ikeda, was selected for further testing for bovine haemoplasmas. Of these samples, 6/47 (13%) and 13/47(28%) were positive for M. wenyonii and Candidatus M. haemobos, respectively. There was no difference in the proportion of samples positive for the bovine haemaplasmas between cattle with anaemia that were negative for T. orientalis (6/20, 33%), or without anaemia or T. orientalis (10/18, 56%), or from cattle herds experiencing anaemia and infection with T. orientalis Ikeda type (3/9, 33%).

DIAGNOSIS: Bovine haemoplasmosis.

CLINICAL RELEVANCE: The presence of bovine haemoplasmas in blood does not establish causality for anaemia in cattle. Diagnosis of anaemia associated with haemoplasmosis would require exclusion of other causes of regenerative anaemia and an association of the agent with anaemia in affected cattle herds. The data collected in this study did not provide evidence that bovine haemoplasmas were associated with a large number of outbreaks of anaemia in cattle in New Zealand.     

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.     

Ovine keratoconjunctivitis experimentally induced by instillation of Mycoplasma conjunctivae

Summary

Five sheep, free from Mycoplasma conjunctivae and ocular Chlamydia infection, were experimentally inoculated with M. conjunctivae and five more sheep were exposed to the infection by contact. Keratoconjunctivitis developed in all ten sheep. As in natural outbreaks of infectious keratoconjunctivitis (IKC), clinical signs were generally moderate and transient, and recurred in some sheep. M. conjunctivae was detected throughout the 53‐day observation period. Clinical diagnosis was confirmed by histopathologic examination of three sheep. Moraxella ovis, found in six of the ten sheep before the start of the experiment, appeared to play no etiologic role in the development of IKC.     

Pathology of Brucella ovis infection in red deer stags (Cervus elaphus)

Abstract

AIM: To describe the pathology of the reproductive tract of red deer stags with active Brucella ovis infection and in stags in which B. ovis infection had resolved.

METHODS: Twenty-three red deer stags of varying history were slaughtered and their epididymides and accessory sex glands examined grossly and by histopathology. At the time of slaughter five of the stags had an active B. ovis infection of 24–55 days duration following exposure to infected rams, 10 stags had been experimentally infected with B. ovis by intravenous inoculation 649 days previously and had developed an active infection but the bacterial infection had resolved at least 308 days prior to slaughter, and eight stags had not been exposed to B. ovis at any time.

RESULTS: Of the five stags with an active infection, one had gross enlargement of the epididymides that could be detected by scrotal palpation. Histological lesions in all five stags included mild to severe, predominantly non-suppurative epididymitis, vesiculitis, prostatitis and ampullitis, with neutrophil exudation in associated glandular ducts. Additional lesions in the epididymides were spermatic granulomas and epithelial hyperplasia with intra-epithelial cyst formation. Of the 10 stags in which the bacterial infection had resolved, two had gross enlargement of the epididymides. The histological lesions were similar to those in stags with active infection but were generally milder, with increased periductal scar tissue in the epididymides. The lesions seen in stags resembled those seen in rams with B. ovis infection but they were usually less florid and had fewer plasma cells. No gross abnormalities or histopathological lesions were detected in the non-infected stags.

CONCLUSIONS: Only a small percentage of red deer stags infected with B. ovis develop lesions of epididymitis that can be detected by scrotal palpation. Gross and histological lesions of the genital tract of stags associated with B. ovis infection are similar to the lesions seen in rams. Lesions in stags persist for >300 days after the bacterial infection has resolved.

CLINICAL RELEVANCE: Brucella ovis infection should be considered when there are gross lesions of epididymitis or histological evidence of inflammation in the epididymides or accessory sex glands of red deer stags. Retrospective diagnosis of B. ovis in stags could be achieved by histological examination of the reproductive organs.     

A real-time polymerase chain reaction assay for the detection of Mycoplasma agalactiae

AIM: To develop a real-time PCR for the detection of Mycoplasma agalactiae, using PCR primers targeting the ma-mp81 gene.

METHODS: A group of 15 M. agalactiae isolates, 21 other Mycoplasma spp. isolates and 21 other bacterial isolates was used in evaluation of the assay.

RESULTS: All M. agalactiae isolates were detected by the assay and none of the non-target isolates was amplified. The analytical detection limit of the assay was 10 fg of purified genomic DNA and 10⁴ cfu/ml milk inoculated with M. agalactiae. When applied to goat-milk samples collected from three herds free of M. agalactiae infection, the assay had a specificity of 100%.

CONCLUSIONS: The assay would be useful in a diagnostic laboratory, providing specific, sensitive and rapid detection of M. agalactiae.     

A feline hemoplasma, 'Candidatus Mycoplasma haemominutum', detected in dog in Japan

We examined for 'Candidatus Mycoplasma haemominutum' infection in 167 blood samples collected from domestic dogs between 2008 and 2009 in the Tohoku area, Japan, and found 5 (3.0%) were positive by PCR assay. This is the first demonstration of 'Candidatus Mycoplasma haemominutum', a feline haemotropic mycoplasma, in the dogs raised 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.     

Real-time quantitative polymerase chain reaction detection of haemoplasmas in healthy and unhealthy dogs from Central Macedonia, Greece

Objectives : The aims of this study were to determine the prevalence of canine haemoplasmas, Mycoplasma haemocanis and “Candidatus Mycoplasma haematoparvum” infection in Central Macedonia, Greece, and to evaluate any associations between canine haemoplasma infection and clinical presentation, selected laboratory data or the presence of ticks. Methods : Genomic DNA was purified from excess blood (n=151) submitted for haematological examination. Purified DNA was subjected to species‐specific quantitative polymerase chain reaction assays duplexed with a canine DNA control quantitative polymerase chain reaction. Clinical records were retrospectively examined and selected clinical parameters were compared to haemoplasma infection status. Results : Nine samples were excluded due to inadequate canine DNA polymerase chain reaction results. Of the remaining 142 samples: eight (5·6%) were positive for M. haemocanis alone, six (4·2%) were positive for “Ca. M. haematoparvum” alone and one (0·7%) was dual positive. No association was found between haemoplasma status and age, sex, breed, health status, presence of anaemia, selected biochemistry parameters, presence of ectoparasites, routine ectoparasiticide treatment or the presence of selected tick‐borne diseases.     

Detection of a novel hemoplasma based on 16S rRNA gene DNA in captive and free-ranging capybaras (Hydrochaeris hydrochaeris)

Two different species of hemoplasmas, Mycoplasma coccoides and M. haemomuris, are known to infect small rodents such as mice and rats. However, there are no previous reports of hemoplasma infection in capybara (Hydrochaeris hydrochaeris). The aim of our study was to determine whether these hemoplasmas might infect capybaras from Southern Brazil. Blood samples from 31 animals: 10 captive and 21 free-ranging capybaras were collected and packed cell volume and total plasma protein were measured. DNA was extracted and PCR assays for M. coccoides and M. haemomuris were performed. Using the M. coccoides-PCR assay 64% of the capybaras were positive, 80% free-ranging and 30% from captive animals. The prevalence of infection between the groups was significantly different (p = 0.001). Sequencing of the nearly entire 16S rRNA gene from the positive samples suggested a novel hemoplasma isolate with identity of 92% with M. coccoides and 86% with M. haemomuris. All capybara samples were negative for M. haemomuris infection. DNA of a housekeeping gene was successfully amplified from all samples. This is the first evidence of a hemoplasma infection in capybaras.     

Ectoparasites are the major causes of various types of skin lesions in small ruminants in Ethiopia

Ectoparasites are the major causes of skin lesions in animals. Clinical, skin scraping examination, and histopathological studies were conducted to identify and characterize skin lesions in small ruminants caused by ectoparasites. Mange mites, lice, sheep keds, and ticks were collected from the skin of affected animals for species identification. Skin biopsies were collected from affected part of the skin and fixed in 10% neutral buffered formalin for histopathology. Of 1,000 sheep and 600 goats examined, 815 (81.50%) sheep and 327 (54.5%) goats were infested with one or more types of ectoparasites. Sarcoptes scabiei var ovis, Demodex ovis, Psoroptes ovis, Bovicola ovis, Melophagus ovinus, and Amblyomma variegatum and other tick species were identified from sheep. S. scabiei var caprae, Demodex caprae, Linognathus stenopsis, and A. variegatum and other tick species were identified from goats. Gross skin lesions or defects observed on the skin include stained and ragged wool, loss of wool/hair, nodules, crusts, lichenification, and fissuring. Microscopic evaluation of H and E stained skin sections revealed lesions in the epidermal layer such as hyperkeratosis, acanthosis, and melanin inconsistency on the basal cells of the epidermis. Follicular keratosis, perifolliculitis, frunculosis, perivasculitis, and aggregates of inflammatory cells (of acute and chronic type) with fibrosis were experiential in the dermal layer of the skin. Most of the skin lesions caused by ectoparasites are overlapping. Thus, ectoparasites control program should be executed to reduce skin lesions as skins are the major export commodity of the country.     

Moniezia infection in the dwarf breeds of small ruminants in Southern Nigeria

A survey of Moniezia infection was conducted among two groups of sheep and goats, of dwarf breeds, in Nigeria: a. those being reared under a traditional, extensive husbandry system in groups of rural villages situated in two different ecological zones; and

b. those reared under an intensive system on experimental stations in two ecological zones of Nigeria.

Moniezia expansa was the predominant species encountered in the animals. The incidence of Moniezia infection was higher in sheep than in goats. The highest infection rates were found in kids and lambs younger than eight months old.

The clinical significance of the infection and some of the highlights of the results are discussed. It is concluded that Moniezia infection in small ruminants can pose a problem deserving of more attention and a suggestion is made for studies on the bionomics of the mite forming the intermediate host in Nigeria.     

Leptospirosis as the most frequent infectious disease impairing productivity in small ruminants in Rio de Janeiro,Brazil

Despite the importance of small ruminants breeding in developing countries, milk/meat productivity remains unsatisfactory. Infectious diseases, such as leptospirosis, brucellosis, and small ruminant lentiviruses (SRLVs), contribute to this scenario. The objective of the present study was to determine the role of each of these diseases in the productivity of small ruminants breeding in Rio de Janeiro, Brazil. In goats, 343 samples were tested for leptospirosis, 560 for Brucella abortus, and 506 for caprine arthritis-encephalitis (CAE), whereas in sheep, 308 samples were tested for leptospirosis, 319 for B. abortus, 374 for Brucella ovis, and 278 for Maedi-Visna (MV). Regarding leptospirosis, 25.9% of goats and 47.4% sheep were seroreactive, with serovar Hardjo the most prevalent in both species. Anti-B. abortus agglutinins were found in 0.7% of all samples, exclusively in goats. In relation to SRLVs, 8.6% of goats and 3.2% of sheep samples were positive for CAE and MV, respectively. Leptospirosis was the major infectious problem in the small ruminants sampled and may contribute to impaired productivity of these animals.     

The occurrence of the feline “Candidatus Mycoplasma haemominutum” in dog in China confirmed by sequence-based analysis of ribosomal DNA

In the present study, polymerase chain reaction (PCR) assay was used to detect haemoplasmas (haemotropic bacteria) in 40 clinically healthy pet dogs in Foshan city, Guangdong Province, China, and one dog was found positive. Comparison of its 16S ribosomal DNA (rDNA) sequence with relevant sequences showed that the isolated haemoplasma had greater sequence identity to feline species “Candidatus Mycoplasma haemominutum” (99%) than to “Candidatus Mycoplasma haematoparvum” (95%). This result, for the first time, indicates the presence of the feline “Candidatus Mycoplasma haemominutum” in Chinese dogs and it represents the first survey of its kind in China by using PCR assay. The results indicated that dog may represent one of the hosts for the feline “Candidatus Mycoplasma haemominutum”.     

Pelvic dimensions of the romney ewe

A gel diffusion test with sonicated Brucella ovis antigen and an enzyme-linked immunosorbent assay based on heat-extracted antigen were used to distinguish false from true reactions in a complement fixation test based on heat-extracted antigen. Of 142 complement fixing reactors (occurring in supposedly Brucellu ovis-free, accredited flocks), the gel diffusion test correctly identified the status of 139 animals as compared to 128 with the enzyme-linked immunosorbent assay. A combination of the two methods resulted in a correct identification of 141 animals. The procedures provide an easy, cheap and quick way to determine the true status of reactors that show up during routine use of the complement fixation test in Brucella ovis re-accreditation procedures.     

Investigation of the index case herd and identification of the genotypes of Theileria orientalis associated with outbreaks of bovine anaemia in New Zealand in 2012

CASE HISTORY AND CLINICAL FINDINGS: On 7 September 2012 the Ministry for Primary Industries was notified of a dairy cow with regenerative anaemia (haematocrit (HCT) 0.08?L/L) in a herd of 465 Jersey-Friesian cross cows (index case herd) in the Northland region of New Zealand. Organisms consistent with Theileria spp. were present in red blood cells on a blood smear. No other causes of anaemia were detected following examination of affected cows. Blood samples collected from 29 randomly selected cows on 26 September 2012 showed that 24 (83%) were anaemic (HCT≤0.24 L/L) and therefore fitted the case definition for bovine anaemia associated with Theileria orientalis infection.

LABORATORY FINDINGS: Using a T. orientalis type-specific PCR assay that targeted the single subunit rRNA gene, all of six animals tested were positive for T. orientalis type Ikeda. Blood samples collected from clinically affected cattle in 11 subsequent outbreaks from throughout the North Island showed that T. orientalis Ikeda type was a common finding, but mixed infections with Chitose type were also identified. In addition, using a PCR assay that targeted the major piroplasm surface gene, T. orientalis type 5 was detected in one cow from the Waikato region.

DIAGNOSIS: The presence of T. orientalis type Ikeda, as well as type 5, was confirmed in cattle from outbreaks of bovine anaemia in herds throughout the North Island of New Zealand.

CLINICAL RELEVANCE: Two new types of T. orientalis were identified in this investigation, that were associated with a sudden rise in cases of bovine anaemia. The body of evidence showed that the Ikeda type was implicated as the cause of disease observed in this epidemic.     

Dynamics of <Emphasis Type="Italic">Mycoplasma hyopneumoniae</Emphasis> seroconversion and infection in pigs in the three main production systems

In this study, we investigated the dynamics of Mycoplasma hyopneumoniae infections in 66 pig farms, with different production systems (one-, two-, and three-site systems), and considered different risk factors. Serological assay was used to detect serum antibodies against M. hyopneumoniae and real time polymerase chain reaction (RT–PCR) was performed to detect M. hyopneumoniae DNA in tracheobronchial swabs. Results demonstrated that M. hyopneumoniae infection status was predominantly influenced by the age of the animals and the type of production system. Infection rates were higher in older animals and the prevalence was higher in the one- and two-site systems than in the three-site systems. Dynamics of infection by RT-PCR showed that earlier M. hyopneumoniae infection on one-site farms occurs earlier, while on two- and three-site farms occurs later but spreads faster, suggesting that contact between animals of different age favors the transmission.     

First morphological characterization of 'Candidatus Mycoplasma turicensis' using electron microscopy

At least three haemotropic mycoplasmas have been recognized in cats: Mycoplasma haemofelis (Mhf), 'Candidatus Mycoplasma haemominutum' (CMhm) and 'Candidatus M. turicensis' (CMt). The latter was originally identified in a Swiss pet cat with haemolytic anaemia and shown to be prevalent in domestic cats and wild felids worldwide using molecular methods. So far, there has been no confirmatory morphological evidence of the existence of CMt presumably due to low blood loads during infection while CMhm has only been characterized by light microscopy with discrepant results. This study aimed to provide for the first time electron microscopic characteristics of CMt and CMhm and to compare them to Mhf. Blood samples from cats experimentally infected with CMt, CMhm and Mhf were used to determine copy numbers in blood by real-time PCR and for transmission and scanning electron microscopy. High resolution scanning electron microscopy revealed CMt and CMhm to be discoid-shaped organisms of 0.3 μm in diameter attached to red blood cells (RBCs). In transmission electron microscopy of CMt, an oval organism of about 0.25 μm with several intracellular electron dense structures was identified close to the surface of a RBC. CMhm and CMt exhibited similar morphology to Mhf but had a smaller diameter. This is the first study to provide morphological evidence of CMt thereby confirming its status as a distinct haemoplasma species, and to present electron microscopic features of CMhm.