Brucellosis is a highly infectious disease which induces significant economic losses to the livestock industry by causing abortion and production losses in ruminants. Additionally, Brucella has zoonotic potential to cause Malta fever in humans. Brucella has many species which infect their particular hosts. However mix livestock farming and sharing of same pasture may enhance cross-species transmission to non-preferred hosts. Cross-transmission of Brucella species to peripheral hosts greatly complicates the diagnosis of brucellosis in both animals and humans. There are many risk factors involved in brucellosis. Among these factors ectoparasites, particularly ticks, are important vectors that haven’t received much scrutiny from epidemiologists investigating this disease. Ticks belong to class Arachnida and subclass Acarina. Ticks harbor uncountable microbes in their gut and as a clade transmit bacterial, viral, and protozoal pathogens to animals and humans; these pathogens are referred to as tick-borne diseases (TBD). Brucella is one of the TBD which has been reported previously in many countries, in Pakistan, however, no investigations have been conducted regarding brucellosis in ticks. In view of the economic importance of brucellosis, the present study was designed to investigate the inter-species transmission of brucellosis in nonpreferred hosts using molecular-based tests, comparative evaluation of molecular techniques and preferred clinical specimens for diagnosis of brucellosis, to investigate the role of ticks as vectors of brucellosis and histopathological investigations of skin and hides of tick infested ruminants. In present study blood, serum and ticks were collected from 692 tick infested cattle, 798 buffalo, 471 sheep and 960 goats having a history of abortions in a farm, sharing of same pasture, close contact and mixed farming of small and large ruminants. All serum samples were subjected to screening with Rose-Bengal Plate test. After screening with RBPT the seropositive serum samples were subjected to duplex conventional and real-time PCR for diagnosis of brucellosis, crosstransmission of Brucella species, and comparative evaluation of real-time PCR with conventional PCR. The blood samples of respective seropositive samples were subjected to real-time PCR for comparative evaluation of serum with the blood for a preferred specimen. Seronegative samples were also diagnosed by real-time PCR assay to investigate the role of non-reactive ruminants in brucellosis. Ticks harvested from real-time PCR positive ruminants were identified; female ticks were subjected to real-time PCR assay. The tissue samples of naturally tick infested skins and hides were studied at the microscopic level. We uncovered cross-species transmission of B. abortus in caprine and ovine serum samples while B. melitensis DNA traces were detected in bovine serum samples. Brucellosis was detected in seronegative small and large ruminants. We also developed and tested a real-time PCR assay more sensitive than conventional PCR and established that brucellosis detection was more accurate when serum samples were used rather than whole blood. The presence of DNA from several Brucella species were detected in ticks using real-time PCR assay. Histopathological examination showed ticks cause significant damage to skin and hides by inducing degenration of the epidermal layer from basal layer, collagen degeneration with a focal area of necrosis, adjacent subdermal abscess and infiltration of neutrophils. Control of ticks should be given consideration to reduce the severity of hide damage and concomitant losses in the domestic leather industry. Ticks are known as a vector of numerous pathogens; efforts are underway to educate farmers about financial loss of skin and hide due to tick infestation and preventive control measures