The role of sheep (Ovis aries) in maintaining Theileria orientalis Ikeda type infection.

Theileria orientalis is a tick‒borne intracellular parasite of red blood cells that causes severe and mild infections in various ruminants worldwide. To date there have been 11 types identified within this species, of which 4 types are presently found in New Zealand cattle. Since 2012, New Zealand has suffered a substantial epidemic of infectious bovine anaemia in both dairy and beef cattle associated with the Ikeda type. The speed at which the disease spread through the North Island suggested that other species could have been involved in transmission. The aim of a series of related experiments was to test the null hypothesis that sheep cannot maintain T. orientalis Ikeda type infection or infect ticks that feed on them. Several studies were conducted over 2 years to address this hypothesis which together showed that sheep can have detectable levels of T. orientalis Ikeda type infection in both the acute and chronic phase and that Haemaphysalis longicornis larvae can become infected when feeding on sheep. No anaemia, weight loss or clinical disease was recorded in the sheep in the acute phase of infection. The levels of infection recorded in the sheep were much lower than those found in cattle, consistent with the sheep being asymptomatic carriers of T. orientalis Ikeda type infection.

Pathogenesis of liver lesions in Theileria orientalis-inoculated cattle and severe combined immunodeficiency mice with bovine erythrocyte transfusion.

Theileria orientalis (T. orientalis) is a bovine protozoal disease similar to malaria in humans. Although the common outcome of malaria in humans and T. orientalis infection in cattle is hepatic disorder, the mechanisms of its development remain unknown. In this study, we investigated hepatocyte injury characterized by accumulation of macrophages with ingested erythrocytes in sinusoid and extramedullary hematopoiesis in cattle and mice experimentally infected with T. orientalis (T. orientalis-infected cattle and T. orientalis-infected mice). Vacuolization of hepatic cells was frequently observed in the vicinity of the aggregated macrophages in the liver sinusoids of T. orientalis-infected mice. A significant percentage of the macrophages accumulated in the liver sinusoids of the severely infected cattle and mice (14.6% and 24.2 to 53.2%, respectively) reacted positively with interleukin-1, interleukin-6 and TNF-α antibodies. Increase in the production of these cytokines was confirmed in T. orientalis-infected cattle and mice by real-time RT-PCR. These findings strongly suggest that increased cytokine production by the macrophages that have phagocytosed T. orientalis-infected erythrocytes causes hepatic disorder in T. orientalis-infected animals.

Effects of Theileria orientalis Ikeda type infection on libido and semen quality of bulls.

There is an epidemic in New Zealand of infectious bovine anaemia associated with Theileria orientalis Ikeda type, an obligate intracellular protozoan parasite. To establish whether T. orientalis Ikeda type infection adversely affects fertility of bulls used for natural mating, a randomised controlled experimental study was conducted. Ten of 17 2-year-old Friesian bulls that had not been previously infected with T. orientalis were infected with T. orientalis Ikeda type and then evaluations occurred during a 20-week period. There were semen and libido evaluations every 2 weeks, starting 4 weeks before the date of infection. In addition, there were blood collections, for haematocrit and infection intensity evaluations, rectal temperatures recorded, and bulls weighed three times weekly for 13 weeks after infection and then once weekly until completion of the study. Physical activity meters were also attached from Days 9-60 and 65-124 post-infection. The ten bulls were successfully infected with T. orientalis Ikeda type and this resulted in a decrease in HCT to about 0.25 by 70 days post-infection. There were no effects of infection on semen quality; however, during the acute phase of infection, when the infection intensity was rapidly increasing, the infected bulls took a longer time period for repeated mounting of females, and were less dominant in the herd social heiracrchy. In conclusion, although the transitory effects on libido could reduce conception rates, the overall effects of T. orientalis Ikeda type infection on bull fertility will probably be little.

An appraisal of oriental theileriosis and the Theileria orientalis complex, with an emphasis on diagnosis and genetic characterisation.

Oriental theileriosis, a tick-borne disease of bovids caused by members of the Theileria orientalis complex, has a worldwide distribution. Globally, at least 11 distinct genotypes of T. orientalis complex, including type 1 (chitose), type 2 (ikeda), type 3 (buffeli), types 4 to 8, and N1-N3, have been described based on the sequence of the major piroplasm surface protein (MPSP) gene. Of these 11 genotypes, mainly ikeda and chitose are known to be pathogenic and cause considerable morbidity (including high fever, anaemia, jaundice and abortion), production losses and/or mortality in cattle. Mixed infections with two or more genotypes of T. orientalis is common, but do not always lead to a clinical disease, posing challenges in the diagnosis of asymptomatic or subclinical forms of oriental theileriosis. The diagnosis of oriental theileriosis is usually based on clinical signs, the detection of piroplasms of T. orientalis in blood smears, and/or the use of serological or molecular techniques. This paper reviews current methods used for the diagnosis of T. orientalis infections and the genetic characterisation of members of the T. orientalis complex, and proposes that advanced genomic tools should be established for investigations of these and related haemoparasites.

The Pathology of Pathogenic Theileriosis in African Wild Artiodactyls.

The published literature on schizont-"transforming," or pathogenic theileriosis, in African wild artiodactyls is dated and based on limited information. Here the authors review the taxonomy, diagnosis, epidemiology, hematology, pathology, and aspects of control in various species. Molecular studies based on 18S and 16S rRNA gene sequences have shown that African wild artiodactyls are commonly infected with diverse Theileria spp., as well as nontheilerial hemoprotozoa and rickettsia-like bacteria, and coinfections with pathogenic and nonpathogenic Theileria species are often recorded. Although theileriosis is still confusingly referred to as cytauxzoonosis in many species, the validity of a separate Cytauxzoon genus in artiodactyls is debated. The epidemiology of theileriosis is complex; the likelihood of fatal disease depends on the interplay of parasite, vertebrate host, tick vector, and environmental factors. Roan calves (Hippotragus equinus) and stressed animals of all host species are more susceptible to fatal theileriosis. Even though regenerative anemia is common, peripheral blood piroplasm parasitemia does not correlate with disease severity. Other than anemia, common macroscopic lesions include icterus, hemorrhages (mucosal, serosal, and tissue), fluid effusions into body cavities, lung edema, and variably sized raised cream-colored foci of leukocyte infiltration in multiple organs. Histopathologic findings include vasocentric hyperproliferation and lysis of atypical leukocytes with associated intracellular schizonts, parenchymal necrosis, hemorrhage, thromboembolism, and edema. Immunophenotyping is required to establish the identity of the schizont-transformed leukocytes in wild ungulates. Throughout the review, we propose avenues for future research by comparing existing knowledge on selected aspects of theileriosis in domestic livestock with that in African wild artiodactyls.

Infection dynamics of Theileria equi and Theileria haneyi, a newly discovered apicomplexan of the horse.

Theileria equi infection, exotic to the United States has reemerged through intravenous (iatrogenic) and tick-borne transmission. Surveillance at the US-Mexico border identified a new species, Theileria haneyi, (T. haneyiEP) (EP = Eagle Pass, Texas) which warranted additional investigation due to inability to detect by PCR targeting of T. equi ema-1 and EMA-1-cELISA validated for T. equi. Infection dynamics of T. haneyiEP were evaluated, including ability to superinfect in the presence of T. equi-Texas (T. equiTX), the isolate responsible for the reemergence of T. equi in the U S. Experimental infection with T. equiTX or T. haneyiEP revealed minimal clinical disease however, T. equiTX infection led to significantly greater neutropenia. Comparison of time to antibody detection following inoculation revealed significantly greater time to detectable anti-T. haneyiEP antibody (26.67 days post-inoculation (DPI)) than T. equiTX (11.67 DPI). Regardless of initial infection with either T. equiTX or T. haneyiEP, superinfection was established. Comparative analysis of antibody responses from a splenectomized horse infected with T. haneyiEP to that of a spleen intact horse infected with T. equiFL revealed a different antibody binding profile to T. haneyiEP, T. equiTX and T. equiFL merozoite antigen and limited shared antigen/cross-reactive antibody(s). Affinity purified T. equi EMA-1 and EMA-2 from T. equiFL were shown as targets for horse antibodies against T. haneyi. Data presented here show (1) T. haneyiEP can superinfect in the presence of T. equiTX infection and co-persists for minimally 25 months, (2) intravenous challenge with T. haneyi is subclinical, and (3) limited cross-reactive antibody between T. haneyiEP and T. equi includes reactivity to EMA-1 and EMA-2.

Theileria highjacks JNK2 into a complex with the macroschizont GPI (GlycosylPhosphatidylInositol)-anchored surface protein p104.

Constitutive c-Jun N-terminal kinase (JNK) activity characterizes bovine T and B cells infected with Theileria parva, and B cells and macrophages infected with Theileria annulata. Here, we show that T. annulata infection of macrophages manipulates JNK activation by recruiting JNK2 and not JNK1 to the parasite surface, whereas JNK1 is found predominantly in the host cell nucleus. At the parasite's surface, JNK2 forms a complex with p104, a GPI-(GlycosylPhosphatidylInositol)-anchor T. annulata plasma membrane protein. Sequestration of JNK2 depended on Protein Kinase-A (PKA)-mediated phosphorylation of a JNK-binding motif common to T. parva and a cell penetrating peptide harbouring the conserved p104 JNK-binding motif competitively ablated binding, whereupon liberated JNK2 became ubiquitinated and degraded. Cytosolic sequestration of JNK2 suppressed small mitochondrial ARF-mediated autophagy, whereas it sustained nuclear JNK1 levels, c-Jun phosphorylation, and matrigel traversal. Therefore, T. annulata sequestration of JNK2 contributes to both survival and dissemination of Theileria-transformed macrophages.

Establishment of a mouse-tick infection model for Theileria orientalis and analysis of its transcriptome.

Oriental theileriosis caused by Theileria orientalis is an economically significant disease in cattle farming. The lack of laboratory animal models and in vitro culture systems is a major obstacle in the drive to better understand the biology of this parasite. Notably, research on the sporozoite stage of T. orientalis has rarely been undertaken, although such investigations are of paramount importance for vaccine development based on blocking sporozoite invasion of its host animals. In the present study, we established a mouse-tick infection model for propagating T. orientalis in mice and for producing the sporozoite stage in tick salivary glands. Splenectomized severe combined immunodeficient mice transfused with bovine erythrocytes were infected with T. orientalis. The larval ticks of Haemaphysalis longicornis were then fed on the T. orientalis-infected mice. The piroplasm and sporozoite stages were microscopically observed in the mouse blood and nymphal salivary glands, respectively. The transcriptomics data generated from the piroplasm and sporozoite stages revealed a stage-specific expression pattern for the parasite genes. The mouse-tick infection model and the transcriptomics data it has provided will contribute to a better understanding of T. orientalis biology and will also provide much needed information for the design of effective control measures targeting oriental theileriosis.

Experimental infection of Friesian bulls with Theileria orientalis (Ikeda) and effects on the haematocrit, live weight, rectal temperature and activity.

Since 2012, New Zealand has suffered from an epidemic of infectious bovine anaemia associated with T. orientalis (Ikeda), an obligate intracellular protozoan parasite of cattle. Despite widespread agreement that T. orientalis (Ikeda) infection has impacted beef and dairy farming in New Zealand there is very little quantitative data to support this conclusion. A randomised controlled experimental study of the effect of T. orientalis (Ikeda) infection on the live weight, haematocrit (HCT), temperature and activity of 2-year-old Friesian bulls was conducted at a Massey University Research farm, Palmerston North. Ten out of seventeen 2-year-old Friesian bulls were injected intravenously with 30 mL whole blood from 2 clinical cases of Theileria-associated bovine anaemia and then followed over a period of 20 weeks. The bulls were blood sampled, had rectal temperature recorded and weighed 3 times weekly for 13 weeks and then once weekly thereafter until the end of the trial. Infection intensity was monitored using qPCR. All 10 inoculated bulls were successfully infected with T. orientalis (Ikeda). The results showed that the live weight response to infection was varied and the bulls could be divided into two groups based on this response. Four infected bulls showed a significant weight reduction of 41.5 kg (p < 0.0001), a financial loss of around NZ$112 per bull, compared with the other 6 bulls in the infected group, which were not different to the 7 uninfected controls. The live weight of the 4 poor growing bulls was significantly lower than the other 6 infected bulls from Day 71 post infection (p < 0.05). All ten infected bulls showed a similar decrease in HCT, with the lowest HCT reached around Day 60 to 80 post-infection, however the four infected bulls that grew poorly did have a significantly elevated HCT for the first 1 to 3 weeks post infection (p < 0.05). The 4 infected bulls which grew poorly also had a significantly higher infection intensity than the other infected bulls from Day 27 to Day 60 post-infection (p < 0.05). There was no pyrexia recorded in the infected group or control groups, instead there was a tendency for the infected group to have a lower rectal temperature from Day 5 to 70 post infection. The infected bulls walked on average 239 steps per day less than the control bulls, although this difference was not significant (p = 0.35). Overall the study clearly showed, by controlling infection date and infectious dose, that a proportion of cattle infected with T. orientalis (Ikeda) have significantly decreased live weight gains.

The diagnostic importance of coagulation parameters in cattle having natural theileriosis.

The purpose of this study was to determine the diagnostic importance of coagulation parameters in cattle with natural theileriosis. Nine Holstein cross-breed cattle with theileriosis as infected group and 6 healthy Holstein cattle as control group were used in the present study. Mean fibrinogen level, thrombin time (TT), activated partial thromboplastin time (aPTT) and prothrombin time (PT) were not statistically different when control and infected groups compared, except for the D-dimer concentration. Quantitative D-dimer concentrations were determined by immune-turbidimetric assay. D-dimer values increased significantly (p<0.05) in infected group (631.55 ± 74.41 µg/L) compared to control group (370.00 ± 59.94 µg/L). D-dimer sensitivity and specificity were also determined at cut-off concentrations (372 µg/L). Sensitivity and specificity of D-dimer values were determined to be 88.89% and 83.33%, respectively. D-dimer is thought to be important indicator in the evaluation of the prognosis in theileriosis cases. Analysis of D-dimer values before and after treatment in controlled case studies were suggested in future studies to enlighten the issue.

PKA and Apicomplexan Parasite Diseases.

The cAMP-dependent protein kinase PKA is a well-characterized member of the serine-threonine protein AGC kinase family and is the effector kinase of cAMP signaling. As such, PKA is involved in the control of a wide variety of cellular processes including metabolism, cell growth, gene expression and apoptosis. cAMP-dependent PKA signaling pathways play important roles during infection and virulence of various pathogens. Since fluxes in cAMP are involved in multiple intracellular functions, a variety of different pathological infectious processes can be affected by PKA signaling pathways. Here, we highlight some features of cAMP-PKA signaling that are relevant to Plasmodium falciparum-infection of erythrocytes and present an update on AKAP targeting of PKA in PGE2 signaling via EP4 in Theileria annulata-infection of leukocytes and discuss cAMP-PKA signling in Toxoplasma.

An outbreak of oriental theileriosis in dairy cattle imported to Vietnam from Australia.

This study reports an outbreak of oriental theileriosis in dairy cattle imported to Vietnam from Australia. Following clinical and pathological diagnoses, a total of 112 cattle blood samples were divided into three groups and tested using multiplexed tandem PCR. Group 1 were from aborted heifers in Vietnam; group 2 were from cattle before shipment from group 1 cattle and group 3 were from the same batch of cattle but transported to Taiwan. Theileria orientalis DNA was detected in 72·3% cattle. The prevalences of T. orientalis in groups 1, 2 and 3 were 77·6, 86·9 and 57·5%, respectively, and the difference in prevalence was significant between groups 1 and 3 (P < 0·0001). The infection intensities of genotypes chitose and ikeda of T. orientalis were higher in groups 1 (57 721 and 33 709, respectively) and 3 (5897 and 61 766, respectively) than those in group 2 (2071 and 6331, respectively). Phylogenetic analyses of the major piroplasm surface protein sequences revealed that genotypes chitose and ikeda determined herein were closely related to those previously reported from Australia. This first report of an outbreak of oriental theileriosis in imported cattle emphasizes improved measures for the export and import of cattle infected with T. orientalis.

Dermatological signs in bovine tropical theileriosis (T heileria annulata infection), a review.

Bovine tropical theileriosis (Theileria annulata infection) is an important tick-borne disease in several regions of the world. This paper describes two clinical cases of tropical theileriosis in northern Tunisia with the uncommon sign of skin nodules. The density of nodules was estimated at 15-20 per 10 cm2; they were distributed over the animal's body and were 0.5-2 cm in diameter. Microscopic examination of the skin nodules 18 days after treatment onset showed a perivascular infiltrate of lymphocytes, macrophages and eosinophils. Nevertheless, no schizonts were observed. Appropriate treatment led to the recovery of one of the two animals with total disappearance of the skin lesions; the other animal died. A review of clinical cases of tropical theileriosis (T. annulata infection) with cutaneous lesions is presented. Various dermatological signs are described in the literature: skin nodules, haemorrhagic lesions, cutaneous gangrene, etc. Most authors reported the presence of schizonts in the nodular lesions. Dermatological involvement in bovine tropical theileriosis should be considered by field veterinarians and should be differentiated from other diseases with dermatological signs.

La theilériose tropicale bovine (infection à Theileria annulata) est une maladie majeure à transmission vectorielle présente dans plusieurs régions du globe. Les auteurs décrivent deux cas cliniques de theilériose tropicale survenus dans le nord de la Tunisie, qui présentaient un tableau peu courant de lésions nodulaires. La densité estimée des nodules était de l'ordre de 15 à 20 nodules pour 10 cm² ; ils mesuraient de 0,5 à 2 cm de diamètre et étaient présents sur toutes les parties du corps de l'animal. L'examen au microscope des nodules cutanés 18 jours après le début du traitement a révélé une infiltration périvasculaire de lymphocytes, macrophages et éosinophiles. En revanche, aucun schizonte n'a été observé. L'administration d'un traitement approprié a éliminé l'infection chez l'un des deux animaux, avec une disparition totale de ses lésions cutanées ; l'autre animal est mort. Les auteurs présentent une synthèse des cas cliniques de theilériose tropicale (infection à T. annulata) s'accompagnant de lésions cutanées. Plusieurs manifestations dermatologiques sont décrites dans la littérature : nodules cutanés, lésions hémorragiques, gangrène cutanée, etc. La plupart des auteurs signalent la présence de schizontes dans les lésions nodulaires. Les manifestations dermatologiques associées à la theilériose tropicale bovine doivent être prises en compte par les vétérinaires de terrain et faire l'objet d'un diagnostic différentiel par rapport à d'autres maladies présentant un tableau dermatologique comparable.

La teileriosis tropical bovina (infección por Theileria annulata) es una importante enfermedad transmitida por garrapatas que afecta a varias regiones del mundo. Los autores describen dos casos clínicos de teileriosis tropical diagnosticados en el norte de Túnez que presentaban un síntoma inhabitual: nódulos cutáneos, con una densidad estimada de 15 a 20 nódulos por cada 10 cm2. Los nódulos, distribuidos por todo el cuerpo del animal, tenían un diámetro de entre 0,5 y 2 cm. El examen microscópico de los nódulos a los 18 días de tratamiento reveló una infiltración perivascular de linfocitos, macrófagos y eosinófilos, pero no se observaron esquizontes. La administración del tratamiento apropiado llevó a la recuperación de uno de los dos ejemplares, con desaparición completa de las lesiones cutáneas, mientras que el otro murió. Los autores pasan revista a una serie de casos clínicos de teileriosis tropical (infección por T. annulata) con presencia de lesiones cutáneas. En la bibliografía están descritos varios casos con síntomas dermatológicos: nódulos cutáneos, lesiones hemorrágicas, gangrena cutánea, etc. La mayoría de los autores señalan la presencia de esquizontes en las lesiones nodulares. Los veterinarios que trabajan sobre el terreno deben tener en cuenta que la teileriosis tropical bovina puede acompañarse de afectación cutánea y distinguir esta enfermedad de otras patologías que provocan síntomas dermatológicos.

Detection of Theileria lestoquardi cross infection in cattle with clinical theileriosis in Iran.

Theileriosis caused by Theileria lestoquardi (malignant ovine theileriosis) in sheep and Theileria annulata (tropical theileriosis) in cattle is an important hemoprotozoal tick-borne disease in Iran. Due to major biologic and phylogenic similarities of these two species, this study was carried out to investigate the occurrence of natural infections with T.lestoquardi and T.annulata in cattle with clinical theileriosis in Ahvaz, southwest Iran. Fifty one cattle were selected based on clinical signs of theileriosis and confirmation by microscopic examination of blood smears. Blood samples were collected from each animal and hematologic and microscopic examinations were performed. Theileria piroplasmic forms were detected in all affected cattle. Pale mucous membranes (43.14%), icterus (11.76%) and fever (70.6%) were also observed. PCR-RFLP analysis revealed T. annulata infection in all tested cattle while coinfections with T. lestoquardi were found in two samples (3.92%). All sampled cattle including the two with mixed species Theileria infection were anemic. This is the first report of Theileria species cross infections in cattle with clinical theileriosis in Iran. It can be concluded that cattle can be infected with both pathogenic Theileria species, T. lestoquardi and T. annulata which can be an important issue in the epidemiology and spread of ovine malignant theileriosis.

The pulmonary involvement in Theileria lestoquardi naturally infected sheep.

Malignant Ovine Theileriosis (MOT) caused by Theileria lestoquardi is considered a major constraint for sheep production in many areas of the world including Sudan. Pulmonary oedema is thought to be the main cause of animal death, but the mechanism, the cell types involved and/or the probable cause of this pneumonia has yet to be defined. The present study was carried out to investigate the pulmonary involvement post T. lestoquardi infection and to identify the cell types involved in pneumonia. Apparently healthy sheep were exposed to ticks challenge in T. lestoquardi endemic area. Lungs impression smears and tissue sections for histopathology were processed. At necropsy, fifteen infected sheep revealed severe pneumonia associated with oedema and accumulation of creamy-grayish frothy exudates. The microscopic findings of examined lungs showed emphysema, congestion, collapse and proliferation of immense amount of different kinds of cells. The current study indicates that T. lestoquardi infections are accompanied with remarkable pulmonary involvements and may lead to respiratory failure and death.

East Coast Fever Caused by Theileria parva Is Characterized by Macrophage Activation Associated with Vasculitis and Respiratory Failure.

Respiratory failure and death in East Coast Fever (ECF), a clinical syndrome of African cattle caused by the apicomplexan parasite Theileria parva, has historically been attributed to pulmonary infiltration by infected lymphocytes. However, immunohistochemical staining of tissue from T. parva infected cattle revealed large numbers of CD3- and CD20-negative intralesional mononuclear cells. Due to this finding, we hypothesized that macrophages play an important role in Theileria parva disease pathogenesis. Data presented here demonstrates that terminal ECF in both Holstein and Boran cattle is largely due to multisystemic histiocytic responses and resultant tissue damage. Furthermore, the combination of these histologic changes with the clinical findings, including lymphadenopathy, prolonged pyrexia, multi-lineage leukopenia, and thrombocytopenia is consistent with macrophage activation syndrome. All animals that succumbed to infection exhibited lymphohistiocytic vasculitis of small to medium caliber blood and lymphatic vessels. In pulmonary, lymphoid, splenic and hepatic tissues from Holstein cattle, the majority of intralesional macrophages were positive for CD163, and often expressed large amounts of IL-17. These data define a terminal ECF pathogenesis in which parasite-driven lymphoproliferation leads to secondary systemic macrophage activation syndrome, mononuclear vasculitis, pulmonary edema, respiratory failure and death. The accompanying macrophage phenotype defined by CD163 and IL-17 is presented in the context of this pathogenesis.

Theileria orientalis: a review.

Theileria orientalis (also known historically as T. sergenti and T. buffeli) is responsible for benign or non-transforming theileriosis, and exerts its major effect through erythrocyte destruction. The life cycle of T. orientalis is essentially similar to that of other Theileria species, except that the schizonts do not induce transformation and fatal lymphoproliferation. The pathogenesis of anaemia as a result of infection is not clearly established and may be multifaceted. Clinical signs of weakness, reluctance to walk and abortion are early but non-specific indications of disease, particularly if accompanied by a history of cattle being moved. Physical examination may reveal pallor (pale eyes, vaginal mucosa), pyrexia, and elevated heart and respiratory rates. T. orientalis is an economically important parasite of cattle in New Zealand, Australia and Japan, especially where naïve animals are introduced into an endemic area or in animals under stress. Increased awareness of the risks posed by the parasite is required to enable management practices to be implemented to minimise its impact.

An assessment of the herd-level impact of the Theileria orientalis (Ikeda) epidemic of cattle in New Zealand, 2012-2013: a mixed methods approach.

AIM: To estimate incidence risk, cumulative mortality and case fatality rate within herds affected by bovine anaemia associated with Theileria orientalis infection (TABA), in New Zealand during the early phase of the epidemic (August 2012-September 2013). METHODS: A mixed methods approach was utilised to integrate data from various sources, including a detailed questionnaire carried out on 18 dairy farms which had experienced cases of TABA; a brief telephone survey of an additional 139 case farms; and data extracted from a Ministry for Primary Industries database for a further 42 case farms. The subsequent analysis determined incidence risk, cumulative mortality and case fatality rates for beef and dairy herds. RESULTS: Data were analysed from 196/263 (74%) known case farms at the date of closing the questionnaires. These farms contained 99,505 cattle; 2,847 animals were reported with clinical disease, and a further 590 animals were recorded as having died from TABA. The within-herd incidence risk, cumulative mortality and case fatality rate were consistent between the three data sources, did not differ between beef and dairy herds, and were estimated to be 0.97 (inter-quartile range (IQR) 0.36-2.07)%, 0.23 (IQR 0.00-0.66)% and 16.67 (IQR 0.00-33.33)%, respectively. There was substantial variability in the level of impact, with 22 farms severely affected (incidence risk >5% and cumulative mortality >5%). CONCLUSIONS: The mixed methods approach was effective in dealing with the disparate data sources. The inclusion of the majority of farms known to be affected at the time the questionnaires were performed implies that the information is likely to be representative. The collective outputs of the analyses represent the best estimate available of within-herd measures of disease frequency in the early phase of the epidemic in New Zealand. The limitations of the data imply that their primary application may be to inform the design of subsequent structured observational field studies. CLINICAL RELEVANCE: The results of this study provide information on the impact of TABA on cattle farms during the emergence and early spread of the disease, as well as for generating hypotheses on causal mechanisms and risk factors that may influence the course of disease.