犬貧血相關傳染性疾病：檢測技術及治療策略之突破

Canine anemia related infectious diseases: Breakthrough of diagnostic technology and therapeutic strategies

劉品辰(Pin-Chen Liu)

指導教授 ： 蘇璧伶

國立臺灣大學/獸醫專業學院/獸醫學研究所/博士(2017年)

https://doi.org/10.6342/NTU201704013

摘要

貧血是犬隻在臨床診斷中最常見的血檢異常之一，造成貧血的原因相當的多，然而在臺灣卻很少有關於犬隻貧血原因的調查報導。我們首先收集2008年1月至2012年間於台大動物醫院就診之貧血犬隻，依據年齡已6月齡為界限，將犬隻分為大於6月齡的一般組與幼犬組，分析血液檢查數據與造成貧血之原因，作為未來臨床獸醫師診斷的參考依據。本次研究中貧血犬隻一共3208隻，其中一般組有3174隻，幼犬組有34隻。調查造成貧血的原因，在一般組中無法判讀造成貧血原因的共2037隻，由單一病因所造成的1435隻，由多重病因所造成的602隻。在單一病因所造成的貧血中，腫瘤、傳染性病原與腎臟疾病相關所引起的貧血為常見的貧血原因。在造成非常嚴重貧血的原因中又以傳染性病原相關疾病占59.7%最為重要，進一步分析傳染性疾病的病原，發現吉貝氏焦蟲症(Babesia gibsoni, B. gibsoni)的感染占非常嚴重貧血原因的83.7%，尤其重要。在幼犬組中無法判讀原因的占21隻，只剩下13隻貧血為單一病因所造成。在這幼犬中以傳染性疾病的犬瘟熱為造成幼年犬隻貧血的最主要因素。所以我們發現在台灣造成犬隻貧血的原因主要來自於傳染性病原，大於6月齡犬隻主要貧血的傳染性病原為吉貝氏焦蟲，而在幼犬則以犬瘟熱(Canine distemper, CD)為主。
焦蟲在台灣的感染是造成成年犬隻貧血的常見原因，因此臨床獸醫師必需將此病納入貧血的鑑別診斷。吉貝氏焦蟲主要由壁蝨媒介導致的血液寄生蟲，儘管已是全球越來越重大的傳染病之一，但在近20年間，對於吉貝氏焦蟲的臨床症狀與血檢異常描述卻多依據零星或少數的病例，缺乏大量且系統性的資料。本研究收集2014年1月至2015年12月，於台大動物醫院就診之經核酸診斷確診感染吉貝氏焦蟲之病例一共62例，其中38隻為單純感染吉貝氏焦蟲之病例，另外24隻為感染吉貝氏焦蟲且合併感染其他疾病之病例，為了進一步探討貧血程度的相關性，我們先將2隻患有免疫性溶血性貧血的犬隻排除避免影響血液學分析，在貧血程度上面不論是單獨或合併感染吉貝氏焦蟲並沒有顯著的差異。血液學方面，最常見的異常為貧血(49/60, 81%)與血小板低下(39/60, 65%)，且其中70%會造成犬隻中度至非常嚴重之貧血。生化檢驗方面，最主要的異常包含高球蛋白血症(28/53, 52.8%)、高膽紅素血症(10/28, 35.7%)與肝指數異常 (7/48, 14.6%)。我們也發現犬隻若不規律使用外寄生預防藥物相較於規律預防的犬隻有較高的比例容易感染吉貝氏焦蟲。
長期以來吉貝氏焦蟲的治療對臨床獸醫師相當具有挑戰性，自從發現在吉貝氏焦蟲cytochrome b (CYTb)上第363的核酐酸單點突變，造成其第121個胺基酸從甲硫胺酸(methionine)轉變為異白胺酸(isoleucine)，使蟲體具有atovaquone抗藥性，研究有別於傳統基因定序能更快速鑑別抗藥性蟲株就顯得非常重要。本研究的目的在於建立SimpleProbe®技術應用於區別吉貝氏焦蟲 CYTb之突變基因。SimpleProbe®是一類特殊類型的雜交探針，此探針與靶序列之間的雜交作用越穩定，溶解溫度也就越高，但如出現單點突變會消弱SimpleProbe®探針結合的穩定性，以在CYTb第363核苷酸單點突變做為設計原理，本研究將89個經聚合酶錄銷反應確認被吉貝氏焦蟲感染的臨床血液樣本，經傳統基因定序CYTb基因確認是否為具atovaquone抗藥性之蟲體後，將使用SimpleProbe®技術分析是否具有抗藥性，相互比對以評估此技術鑑別之效能，其敏感性為100% (42/42)，特異性為95.7% (45/47)，根據本研究依SimpleProbe®所建立之技術能有效並快速區別具atovaquone抗藥性之吉貝氏焦蟲。為了進一步了解自然的抗藥蟲株現況，收集2014至2015年自台大動物醫院與流浪動物確診為吉貝氏焦蟲一共95個血液樣本與其犬隻的藥物治療史進行分析，8個血液樣本呈現具atovaquone抗藥性陽性，其中3個血液樣本來自於台大動物醫院就診之犬隻，在這3個血液樣本中只有1隻狗曾經接受過atovquone治療，其餘5隻陽性犬隻則是在流浪犬隻中發現，此研究首次證實台灣已存在且atovaquone抗藥性野生蟲株。本檢測方法在研究上可以監測atovaquone抗藥性吉貝氏焦蟲的分佈與數量，於臨床上個別犬隻的檢驗結果可以提供臨床獸醫師在藥物選擇上的依據。
在台灣犬瘟熱亦為造成幼年犬隻貧血的常見原因之一，犬瘟熱為犬瘟熱病毒所引起之高傳染力與高致死率的疾病。感染後臨床症狀的嚴重程度受病毒的毒力、感染時的年齡、宿主的免疫狀態和環境壓力所影響。臨床上對於犬瘟熱治療方式為給予對症支持治療，目前於小鼠或貂的研究中給予抗血清或單株抗體做為預防性治療，出現不錯的保護作用，增加被動免疫的抗體力價被認為在動物發病後可能是一種有效的治療方式，然而目前並無任何被動免疫治療實際應用於犬瘟熱患犬療效之評估，因此針對豬抗犬瘟熱病毒抗體次單位[Porcine F(ab’)2]於自然感染犬隻效果之評估，我們收集2008年1月至2013年12月至台大動物醫院就診，出現臨床症狀但起始治療時尚未有神經症狀且年齡小於6個月，以RT-nPCR確診為犬瘟熱感染之患犬41隻，根據治療方式之不同分為兩組，進一步分析其治療組合的效果。第1組(共25隻)為接受豬抗犬瘟熱病毒抗體次單位與對症支持治療組，第2組 (共16隻)則為僅接受對症支持治療組。比較兩組中第1組與第2組的存活率分別為76% (19/25)及31.3% (5/16)，有統計上的差異 (P = 0.04)。在降低神經症狀出現的比例上，第1組與第2組出現神經症的比例分別為32%及75%，二組在統計上也有顯著差異(P < 0.05)。本研究之結果證實豬抗犬瘟熱病毒抗體次單位在活體(in vivo)的實際應用成效，未來應該可以進一步應用在瀕臨絕種的其他物種上。

關鍵字

貧血 ； 吉貝氏焦蟲症 ； 臨床特徵 ； SimpleProbe® ； M121I突變 ； 犬瘟熱 ； 被動免疫治療

並列摘要

Anemia is a common hematologic abnormality in dogs, however, few data are available regarding epidemiology and causes in Taiwan. To investigate the causes of anemia [hematocrit (Hct) < 37%] in dogs, 3208 anemic cases collected between January 2008 and December 2012 at National Taiwan University Veterinary Hospital (NTUVH) were analyzed. Dogs under the age of six months were further collected as puppy group (Hct <22%). In dogs over the age of six months group, among the 2037 dogs with identifiable causes, 70.4% (1435 dogs) were induced by single cause, whereas 29.6% (602 dogs) by multiple causes. Cancer-related anemia (CRA, 460/1435, 32.1%), infectious pathogen-related anemia (IPRA, 287/1435, 20.0%) and renal disease-related anemia (251/1435, 17.6%) are the frequent causes anemia in dogs. Furthermore, IPRA (59.7%) is the primarily result the very severe anemia. Of the 43 infectious disease-related very severe anemic dogs, the most commonly diagnosed pathogen was Babesia gibsoni (B. gibsoni; 83.7%, n = 36). In puppy group, among the 34 dogs examined, the causes of anemia could not be identified in 21 (61.7%) animals. For the remaining dogs, 13 dogs were classified as anemic with a single cause. Canine distemper virus is the most important pathogen causing anemia in puppies and the mortality rate was 100% in this study. Taken together, infectious disease is one of the frequent causes of canine anemia in Taiwan. B. gibsoni and canine distemper appeared to be the most important infectious pathogen causing anemia in dogs over 6 months and puppies, respectively.
B. gibsoni is increasingly recognizes as a cause of canine tick-borne disease in worldwide. However, only a few clinical characteristics and laboratory findings regarding naturally infected B. gibsoni were available. Most of the published reports were mixed with a distinct species, i.e. B. conradae. 62 dogs, with full medical records and positive B. gibsoni PCR results, from January 2014 to December 2015 presented at National Taiwan University Veterinary Hospital (NTUVH), were enrolled in this study. Of 62 dogs recruited, 24 (38.7%) had concurrent disease, 38 (61.3%) had B. gibsoni infection alone. Two dogs were excluded because of concurrent with immune mediated hemolytic anemia, which may dramatically impact the anemic severity. The severity of anemia between B. gibsoni infected and concurrent disease groups showed no significant different (P > 0.05). The common observed hematological abnormality were anemia (49/60, 81.7%) and thrombocytopenia (39/60, 65%). 42 of 35 dogs (70.0%) had moderate to very severe anemia. The main biochemical abnormalities were included hyperglobulinemia (28/53, 52.8%), hyperbilirubinemia (10/28, 35.7%), and elevated hepatic enzyme activities (7/48, 14.6%). Dogs that didn’t regularly received ectoparasidal agents had higher infection rate of B. gibsoni infection.
Several drug combinations have been used to treat B. gibsoni infection. A combination of atovaquone and azithromycin has been widely used throughout the world, particularly in areas with epidemics. However, the drug-resistant atovaquone strain is known to be influenced by various mutations, particularly the substitution of methionine with isoleucine (M121I) in the B. gibsoni cytochrome b (CYTb) gene. Rapid identification of the drug-resistant strain is required to select a more effective combination of drugs. A SimpleProbe® real-time PCR assay was designed to detect the single nucleotide polymorphism at nucleotide 363 in CYTb gene of B. gibsoni and the sensitivity and specificity were evaluated by comparing the results from the conventional DNA sequencing method. When detecting the M121I mutation, 42 of 42 mutant samples were identified, with a sensitivity of 100%, and 45 of 47 wild type samples were identified, with a specificity of 95.7%. Furthermore, we use the SimpleProbe® assay to investigate the naturally ATV-resistant strain of B. gibsoni in Taiwan. 95 dog blood samples confirmed with B. gibsoni infection by PCR method from January 2014 to December 2015. Of all 95 samples, 52 samples were collected from the client-owned dogs in NTUVH and 33 samples were collected from the free-roaming dogs. 3 of client-owned dogs (3/52, 5.8%) and 5 of free roaming dogs (5/33, 15.2%) were detected the M121I mutation. Only one of them had ever been treated with AA combinations. This is the first report that naturally atovaquone-resistant B. gbisoni strain existed in Taiwan.
Canine distemper (CD), caused by a morbillivirus of the family Paramyxoviridae, occurs worldwide and is one of the most contagious and lethal diseases especially in juvenile dogs. The clinical feasibility of passive immunotherapy has not been demonstrated in dogs naturally infected with canine distemper. In this study, porcine anti-canine distemper virus IgG and F(ab’ )2 antibody fragments were used to treat infected puppies. A total of 41 naturally infected puppies (age < six months) exhibiting severe respiratory signs, but lacking neurological signs, were enrolled in the study. Twenty-five puppies were treated with a combination of IgG or F(ab’ )2 antibody fragments (Group 1) and supportive therapy and 16 puppies received routine supportive care only (Group 2). The survival rate of dogs in Group 1 (19/25; 76%) was significantly higher than that in Group 2 (5/16; 31·3%) (P < 0.05). During the therapy, 8 of the 25 dogs (32%) in Group 1 developed neurological signs versus 12 of the 16 dogs (75%) in Group 2 (P < 0.05). Adverse reactions were limited to elevated body temperature in dogs that received IgG antibodies. Porcine anti-canine distemper virus antibodies improved survival in puppies affected with canine distemper with minimal adverse effects. Therefore, this therapy could be considered for treatment of endangered animal species infected with canine distemper virus.

並列關鍵字

Anemia ； canine distemper ； passive immunotherapy ； Babesia gibsoni ； clinical profile ； SimpleProbe® assay ； M121I mutation

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